Multiplex amplification and detection

ABSTRACT

The invention relates to the field of multiplex amplification. In particular, the invention relates to methods for assaying a sample for one or more nucleic acid targets in a single reaction based on the distinct melting temperatures or melting profiles of primers and/or probes. The invention also provides probes and kits for use in such methods.

The present invention relates to the field of multiplex detection. Inparticular, the invention relates to methods for assaying a sample forone or more nucleic acid targets in a single reaction based on thedistinct melting temperatures or melting profiles of probes. Theinvention also provides probes and kits for use in such methods.

Multiplex PCR, which uses multiple pairs of primers to simultaneouslyamplify multiple target sequences in a single PCR reaction, is a moreefficient approach to PCR than standard single primer-pair PCR. Thesimultaneous amplification of various targets reduces both the cost andturn-around time of PCR analysis, minimizes experimental variations andthe risk of cross-contamination, and increases the reliability of endresults. Multiplex PCR has been used in many areas of DNA testingincluding identification of micro-organisms, gene expression analysis,mutation and polymorphism analysis, genotyping and DNA array analysis,and RNA detection.

Real-time PCR has been developed to quantify amplified products duringPCR reactions. Real-time PCR is based on the principles that emission offluorescence from dyes directly or indirectly associated with theformation of newly-synthesized amplicons or the annealing of primerswith DNA templates can be detected and is proportional to the amount ofamplicons in each PCR cycle. Real-time PCR is carried out in aclosed-tube format and it is quantitative. Several methods are currentlyavailable for performing real-time PCR, such as utilising TaqMan probes(U.S. Pat. Nos. 5,210,015 and 5,487,972, and Lee et al., Nucleic AcidsRes. 21:3761-6, 1993), molecular beacons (U.S. Pat. Nos. 5,925,517 and6,103,476, and Tyagi and Kramer, Nat. Biotechnol. 14:303-8, 1996),self-probing amplicons (scorpions) (U.S. Pat. No. 6,326,145, andWhitcombe et al., Nat. Biotechnol. 17:804-7, 1999), Amplisensor (Chen etal., Appl. Environ. Microbiol. 64:4210-6, 1998), Amplifluor (U.S. Pat.No. 6,117,635, and Nazarenko et al., Nucleic Acids Res. 25:2516-21,1997, displacement hybridization probes (Li et al., Nucleic Acids Res.30:E5, 2002); DzyNA-PCR (Todd et al., Clin. Chem. 46:625-30, 2000),fluorescent restriction enzyme detection (Cairns et al. Biochem.Biophys. Res. Commun. 318:684-90, 2004) and adjacent hybridizationprobes (U.S. Pat. No. 6,174,670 and Wittwer et al., Biotechniques22:130-1, 134-8, 1997). Most of these probes consist of a pair of dyes(a reporter dye and an acceptor dye) that are involved in fluorescenceresonance energy transfer (FRET), whereby the acceptor dye quenches theemission of the reporter dye. In general, the fluorescence-labelledprobes increase the specificity of amplicon quantification.

Another form of probe used in PCR is a double-stranded linear probewhich has two complementary oligonucleotides. The probes described inthe prior art have been of equal length, in which at least one of theoligonucleotides acts as a probe for a target sequence in asingle-stranded conformation. The 5′ end of one of the oligonucleotidesis labelled with a fluorophore and the 3′ end of the otheroligonucleotide is labelled with a quencher, e.g., an acceptorfluorophore, or vice versa. When these two oligonucleotides are annealedto each other, the two labels are close to one another, therebyquenching fluorescence. Target nucleic acids, however, compete forbinding to the probe, resulting in a less than proportional increase ofprobe fluorescence with increasing target nucleic acid concentration(Morrison L. et al., Anal. Biochem., Vol. 183, pages 231-244 (1989);U.S. Pat. No. 5,928,862).

Double-stranded linear probes modified by shortening one of the twocomplementary oligonucleotides by a few bases to make a partiallydouble-stranded linear probe, are also known in the art. In suchdouble-stranded linear probes in the prior art, the longeroligonucleotide has been end-labelled with a fluorophore and theslightly shorter oligonucleotide has been end-labelled with a quencher.In the double-stranded form, the probe is less fluorescent due to theclose proximity of the fluorophore and the quencher. In the presence ofa target, however, the shorter quencher oligonucleotide is displaced bythe target. As a result, the longer oligonucleotide (in the form ofprobe-target hybrid) becomes substantially more fluorescent (Li et al.,Nucleic Acids Research, Vol. 30, No. 2, e5 (2002)).

US 2005/0227257 describes a slightly modified double stranded linearnucleic acid probe. The probe described in this patent application ismodified by shortening one of the two complementary oligonucleotides bymore bases, compared to the above, to make a partially double-strandedlinear probe.

Fluorescent hybridisation probes have also been used in other fields.For example, methods for multiplex genotyping using fluorescenthybridisation probes have been described (e.g. U.S. Pat. No. 6,140,054)which use the melting temperature of fluorescent hybridization probesthat hybridize to a PCR amplified targeted region of genome/nucleic acidsequence to identify mutations and polymorphisms.

The advent of high-throughput genetic testing has necessitated bothqualitative and quantitative analysis of multiple genes and has led tothe convergence of multiplex PCR and real-time PCR into multiplexreal-time PCR. Since double-stranded DNA intercalating dyes are notsuitable for multiplexing due to their non-specificity,fluorescence-labelled probes have made multiplex real-time PCR possible.However, multiplex real-time PCR is limited by the availability offluorescence dye combinations. Currently, only up to four or fivefluorescence dyes can be detected and quantified simultaneously inreal-time PCR.

US2005/0053950 describes a protocol for quantifying multiplex real-timepolymerase chain reactions (PCR). The methods quantify multiple PCRproducts or amplicons in a single real-time PCR reaction based on thedifferent melting temperatures (T_(m)) of each amplicon and the emissionchanges of double-stranded DNA dyes such as SYBR Green I when ampliconsare in duplex or in separation. For a specific amplicon with a T_(m),the emission difference between the emission reading taken at atemperature below the T_(m) and the emission reading taken at atemperature above the T_(m) corresponds to the emission value of theamplicon in duplex. Accordingly, the emission difference of eachamplicon in a single PCR reaction can be used to quantify each amplicon.However, the multiplexity and sensitivity of such methods can berelatively low. For example, the difference in melting temperaturesbetween amplicons of the order of 100-150 nucleotides in length issmall. Therefore, these techniques require the use of amplicons withlarge differences in their sizes in order to be able to distinguishbetween them.

There is a need, however, to develop other methods of amplifying andquantifying multiple target sequences in a single PCR reaction formultiplex real-time PCR with greater levels of multiplexity andsensitivity.

The method of the present invention differs from prior art technologies.Firstly, the method is based on the different melting properties (T_(m)or melting profile) of each probe and the emission changes of labels onthe probe when the probe's internal double-stranded portions are induplex or in separation. The probe of present invention comprises adouble-stranded portion which can be formed by a first oligonucleotideand a second oligonucleotide; the double stranded portion has a distinctT_(m) for each probe which distinguishes different probes within a setof probes comprising the same or similar labels. Secondly, the firstoligonucleotide may or may not comprise one or more labels and it is thefirst oligonucleotide which is consumed during the amplificationreaction. The emission difference between the emission readings at twodifferent temperatures corresponds to the emission value of the probeafter some probe being consumed. Thirdly, measuring melting profiles ofunconsumed probes provide an indication of the presence or amount oftarget nucleic acids presented in a sample.

To facilitate understanding of the invention, a number of terms aredefined below.

As used herein, the terms “target sequence”, “target nucleic acid”,“target nucleic acid sequence” and “nucleic acids of interest” are usedinterchangeably and refer to a desired region which is to be eitheramplified, detected or both. The target sequence, which is the object ofamplification and detection, can be any nucleic acid. The targetsequence can be RNA, cDNA, genomic DNA, or DNA or RNA, for example froma disease-causing micro-organism or virus. The target sequence can alsobe DNA treated by chemical reagents, various enzymes and physicalexposure. A target nucleic acid sequence of interest in a sample mayappear as single-stranded DNA or RNA such as cDNA, mRNA, other RNA or asseparated complementary strands. Separating complementary strands oftarget nucleic acid may be accomplished by physical, chemical orenzymatic means. For the ease of description and understanding,references to nucleic acids of interest or targets refer both to thesemoieties as found in a test sample and to amplified copies of portionsof theses nucleic acids, unless specifically noted to the contrary.

“Primer” as used herein refers to an oligonucleotide, whether occurringnaturally or produced synthetically, which is capable of acting as apoint of initiation of synthesis when placed under conditions in whichsynthesis of a primer extension product which is complementary to anucleic acid strand is induced i.e., in the presence of nucleotides andan agent for polymerization such as DNA polymerase and at a suitabletemperature and buffer. The primers herein are selected to besubstantially complementary to the different strands of each specificsequence to be amplified. This means that the primers must besufficiently complementary to hybridize with their respective strands. Anon-complementary nucleotide fragment may be attached to the 5′-end ofthe primer, with the remainder of the primer sequence beingcomplementary to the diagnostic section of the target base sequence.Commonly, the primers are complementary except when non-complementarynucleotides may be present at a predetermined primer terminus asdescribed.

The term “complementary to” is used herein in relation to a nucleotidethat will base pair with another specific nucleotide. Thus adenosine iscomplementary to uridine or thymidine and guanosine is complementary tocytidine. It is appreciated that whilst thymidine and guanosine may basepair under certain circumstances they are not regarded as complementaryfor the purposes of this specification. For purposes of the presentinvention, the term “substantially complementary” means that equal ormore than 70%, preferably more than 80%, more preferably more than 90%and most preferably more than 95% or 99% of nucleobases on one strand ofthe probe finds its Watson-Crick binding partner on the other strand ofthe probe (or in the nucleic acid of interest) in an alignment such thatthe corresponding nucleotides can hybridize to each other.

The terms “duplex” and “double-stranded” are interchangeable, mean onestrand of oligo-poly-nucleotides hybridises to the complementaryoligo-poly-nucleotides.

The term “identical” means that two nucleic acid sequences have the samesequence or a complementary sequence.

As used herein, “continuous monitoring” and similar terms refer tomonitoring multiple times during a cycle of PCR, preferably duringtemperature transitions, and more preferably obtaining at least one datapoint in each temperature transition.

As used herein, “cycle-by-cycle” monitoring means monitoring the PCRreaction once or multiple times each cycle.

The term “Actual Consumed Amount” (ACA) means the amount of probe beingconsumed in a reaction as reflected by fluorescence measuring.

“Amplification” as used herein denotes the use of any amplificationprocedures to increase the concentration of a particular nucleic acidsequence within a mixture of nucleic acid sequences.

The term “sample” as used herein is used in its broadest sense. Abiological sample suspected of containing nucleic acid can comprise, butis not limited to, genomic DNA, cDNA (in solution or bound to a solidsupport), and the like.

The term “label” as used herein refers to any atom or molecule which canbe used to provide or aid to provided a detectable (preferablyquantifiable) signal, and which can be attached to a nucleic acid orprotein. Labels may provide signals detectable by fluorescence,radioactivity, colorimetry, gravimetry, magnetism, enzymatic activityand the like.

The term “adjacent” or “substantially adjacent” as used herein refers tothe positioning of two oligonucleotides on its complementary strand ofthe template nucleic acid. The two template regions hybridised byoligonucleotides may be contiguous, i.e. there is no gap between the twotemplate regions. Alternatively, the two template regions hybridised bythe oligonucleotides may be separated by 1 to about 40 nucleotides, morepreferably, about 1 to 10 nucleotides.

The term “thermally cycling,” “thermal cycling”, “thermal cycles” or“thermal cycle” refers to repeated cycles of temperature changes from atotal denaturing temperature, to an annealing (or hybridising)temperature, to an extension temperature and back to the totaldenaturing temperature. The terms also refer to repeated cycles of adenaturing temperature and an extension temperature, where the annealingand extension temperatures are combined into one temperature. A totaldenaturing temperature unwinds all double stranded fragments into singlestrands. An annealing temperature allows a primer to hybridize or annealto the complementary sequence of a separated strand of a nucleic acidtemplate. The extension temperature allows the synthesis of a nascentDNA strand of the amplicon.

The term “reaction” as used herein refers to hybridisation reaction,extension reaction or amplification reaction or other biological,chemical reactions.

The terms “amplification mixture” or “PCR mixture” as used herein referto a mixture of components necessary to detect target nucleic acid fromnucleic acid templates. The mixture may comprise nucleotides (dNTPs),probes, a thermostable polymerase, primers, and a plurality of nucleicacid templates. The mixture may further comprise a Tris buffer, amonovalent salt, and Mg²⁺. The concentration of each component is wellknown in the art and can be further optimized by an ordinary skilledartisan.

The terms “amplified product” or “amplicon” refers to a fragment of DNAamplified by a polymerase using a pair of primers in an amplificationmethod such as PCR

The term “melting profile” refers to a collection of measurements of anoligo (or poly)nucleotide and its complement which indicate the oligo(or poly)nucleotide molecule's transition from double-stranded tosingle-stranded nucleic acid (or vice-versa). The transition of anucleic acid from double-stranded to single-stranded form is oftendescribed in the art as the “melting” of that nucleic acid molecule. Thetransition may also be described as the “denaturation” or “dissociation”of the nucleic acid. Accordingly, a melting profile of the presentinvention may also be referred to as a “dissociation profile”, a“denaturation profile”, a “melting curve”, a “dissociation curve”, a“hybridisation/dissociation profile” etc.

The “melting temperature” or “T_(m)” of a nucleic acid moleculegenerally refers to the temperature at which a polynucleotidedissociates from its complementary sequence. Generally, the T_(m) may bedefined as the temperature at which one-half of the Watson-Crick basepairs in duplex nucleic acid molecules are broken or dissociated (i.e.,are “melted”) while the other half of the Watson-Crick base pairs remainintact in a double stranded conformation. In preferred embodiments whereduplex nucleic acid molecules are oligonucleotides and in otherembodiments where the duplex nucleic acids dissociate in a two-statefashion, the T_(m) of a nucleic acid may also be defined as thetemperature at which one-half of the nucleic acid molecules in a sampleare in a single-stranded conformation while the other half of thenucleic acid molecules in that sample are in a double-strandedconformation. T_(m), therefore defines a midpoint in the transition fromdouble-stranded to single-stranded nucleic acid molecules (or,conversely, in the transition from single-stranded to double-strandednucleic acid molecules). It is well appreciated in the art that thetransition from double-stranded to single-stranded nucleic acidmolecules does not occur at a single temperature but, rather, over arange of temperatures. Nevertheless, the T_(m) provides a convenientmeasurement for approximating whether nucleic acid molecules in a sampleexist in a single-stranded or double-stranded conformation. As such, themelting temperature of a nucleic acid sample may be readily obtained bysimply evaluating a melting profile for that sample.

The term “consumed” or “consumption” means that the amount of freelabeled probes is decreased at a temperature at which the labeled probeis normally intact. The labeled probe may comprise or not comprisedouble stranded portion; the consumption of the labeled probe may resultin detection signal changes. The consumption of the labeled probe mayinvolve probe hybridisation to target nucleic acid or degradation of theprobe upon hybridisation to target nucleic acid. In the case of theprobe comprising a double stranded portion, the decrease in the amountof free labeled probe may be a result of at least one strand of theprobe being unavailable to form double-stranded portion, which is eitherthe first oligonucleotide of the probe or second oligonucleotide of theprobe or both. The absence of at least one strand of the probe means thefirst oligonucleotide of the probe, the second oligonucleotide of theprobe or both oligonucleotides of the probe are hybridised with thetarget nucleic acid. The hybridisation of the first oligonucleotide ofthe probe, the second oligonucleotide of the probe or botholigonucleotides of the probe with the target nucleic acid may befollowed by extension of the oligonucleotides of the probe which acts asa primer, or by degradation of the oligonucleotides of the probe.

The present invention describes methods that allow the essentiallysimultaneous amplification and detection of a large number of differenttarget nucleic acid sequences.

In a first aspect, the invention provides a method for assaying a samplefor one or more target nucleic acids, said method comprising:

(a) contacting a sample comprising one or more target nucleic acids withan amplification reaction mixture comprising:

-   -   (i) one or more pairs of forward/reverse oligonucleotide        primers, wherein the primer pairs are capable of amplifying one        or more target nucleic acids, if present in the sample,    -   (ii) a set of two or more probes, wherein at least one probe in        the set comprises a double-stranded portion,    -   wherein each probe in the set comprises a detectable label or        detectable combination of labels which is/are capable of        producing a changeable signal which is characteristic for each        probe, and    -   wherein said two or more probes comprise the same detectable        label or different detectable labels with undistinguishable        emission spectra and wherein the melting characteristics of each        of such probes are different, each probe with double-stranded        portion has a signature melting temperature, whereas        single-stranded probes having no double stranded portion does        not have a signature melting temperature;        (b) performing an amplification reaction on the sample/reaction        mixture under amplification conditions, wherein, when a target        nucleic acid is present, the portions of probes which are        substantially complementary to part of that target nucleic acid        are hybridised with the target nucleic acid, therefore being        consumed, wherein the consumption of probes causes changes of        detectable signal in the labels, and the consumed probe are no        longer able to form double stranded portion if the original        probe has a double-stranded portion; and        (c) measuring, at least once, the melting profile of the        unconsumed probes in the reaction mixture by detecting the        signal(s) from the labels in those probes as a function of        temperature, wherein the presence or absence of a melting        characteristics of any probes in the melting profile analysis is        an indication of unconsumption or consumption of that probe,        which further provides an indication of whether or not at least        one target nucleic acid is present in said sample.

In another aspect, the invention provides a method for assaying a samplefor one or more target nucleic acids, said method comprising:

(a) contacting a sample comprising one or more target nucleic acids witha reaction mixture comprising:

-   -   a set of two or more probes, wherein at least one probe in the        set comprises a double-stranded portion,    -   wherein each probe in the set comprises a detectable label or        detectable combination of labels which is/are capable of        producing a changeable signal which is characteristic for each        probe, and    -   wherein said two or more probes comprise the same detectable        label or different detectable labels with undistinguishable        emission spectra and wherein the melting characteristics of each        of such probes are different, each probe with double stranded        portion has a signature melting temperature, whereas        single-stranded probes having no double stranded portion does        not have a signature melting temperature;        (b) performing the reaction on the sample/reaction mixture,        wherein the reaction is a primer extension reaction under        extension conditions, wherein, when a target nucleic acid is        present, the corresponding probe which is extendable, is        hybridised with target nucleic acid, therefore being consumed        during the primer extension reaction, wherein the consumption of        probes causes changes of detectable signal in the labels, and        the consumed probe are no longer able to form double stranded        portion if the original probe has a double-stranded portion; and        (c) measuring, at least once, the melting profile of the        unconsumed probes in the reaction mixture by detecting the        signal(s) from the labels in those probes as a function of        temperature,

wherein the presence or absence of a melting characteristics of anyprobes in the melting profile analysis is an indication of unconsumptionor consumption of that probe, which further provides an indication ofwhether or not at least one target nucleic acid is present in saidsample.

In another aspect, the invention provides a method for assaying a samplefor one or more target nucleic acids, said method comprising:

(a) contacting a sample comprising one or more target nucleic acids witha hybridisation reaction mixture comprising:

-   -   a set of two or more probes, wherein at least one probe in the        set comprises a double-stranded portion,    -   wherein each probe in the set comprises a detectable label or        detectable combination of labels which is/are capable of        producing a changeable signal which is characteristic for each        probe, and    -   wherein said two or more probes comprise the same detectable        label or different detectable labels with undistinguishable        emission spectra and wherein the melting characteristics of each        of such probes are different, each probe with double stranded        portion has a signature melting temperature, whereas        single-stranded probes having no double stranded portion does        not have a signature melting temperature;        (b) performing the hybridisation reaction on the sample/reaction        mixture under hybridisation conditions, wherein, when a target        nucleic acid is present, the corresponding probes which are        substantially complementary to part of that target nucleic acid        are hybridised with target nucleic acid, therefore being        consumed during the reaction, wherein the consumption of probes        causes changes of detectable signal in the labels, and the        consumed probe are no longer able to form double stranded        portion if the original probe has a double-stranded portion; and        (c) measuring, at least once, the melting profile of the        unconsumed probes in the reaction mixture by detecting the        signal(s) from the labels in those probes as a function of        temperature, wherein the presence or absence of a melting        characteristics of any probes in the melting profile analysis is        an indication of unconsumption or consumption of that probe,        which further provides an indication of whether or not at least        one target nucleic acid is present in said sample.

In another embodiment of the present invention, said set of two or moreprobes may comprise at least one single-stranded probe which does notcomprise a double stranded portion.

The single-stranded probe may be a double dye labeled probe which causesdetectable signal changes upon hybridisation of the probe with a targetnucleic acid and/or degradation of the probe.

In another embodiment of the present invention, said set of two or moreprobes may comprise at least two probes having double-stranded portions,

wherein a first probe has a melting temperature T_(m)1 in terms of itsdouble-stranded portion,

wherein a second probe has a melting temperature T_(m)2 in terms of itsdouble-stranded portion,

wherein T_(m)1>T_(m)2,

wherein the same labels are independently attached to the first andsecond probes, wherein a reduction of any melting peak at T_(m)1 and/orT_(m)2 provides indication of consumption of the first and/or secondprobe(s).

In the above mentioned methods, the probe having double-stranded portioncan be molecular beacon probe. Alternatively, the probe havingdouble-stranded portion may comprise:

a first oligonucleotide which comprises a first region and a secondregion, wherein said first region is substantially complementary to partof one target nucleic acid, and

at least one second oligonucleotide which comprises a region which issubstantially complementary to the second region of the firstoligonucleotide,

such that the first and second oligonucleotides are capable of forming adouble-stranded portion.

In any of the above methods, said melting profile may be measured beforereaction/amplification takes place (pre-amplification melting profile),and/or is measured after completion of reaction/amplification(post-amplification melting profile), and/or is measured duringreaction/amplification at each cycle or selected cycles(mid-amplification melting profile),

wherein said method additionally comprises step (d)

(i) comparing at least two melting profiles obtained in (c)and/or(ii) comparing a melting profile obtained in step (c)

with a previously-obtained melting profile of the same probes or

with a melting profile of the same probes obtained in parallel at thesame time in control reactions, or

with a theoretical melting profile of the same probes

wherein a change in the melting profile provides an indication ofwhether or not at least one target nucleic acid is present in saidsample/reaction mixture,

wherein said the pre-amplification melting profile is measured in thesame reaction vessel before the start of reaction/amplification, or ismeasured in a separate reaction vessel where no amplification takesplace due to that reaction mixture lacking one or more ingredientsnecessary for the reaction/amplification,

wherein in step (d) the post-amplification or mid-amplification meltingprofile is compared with the pre-amplification melting profile of theduplex of probes to determine whether a particular probe is consumed,this being indicative of the presence of the corresponding target in thesample.

In another embodiment, the second oligonucleotides of the probes may bephysically separated from the main reaction mix containing firstoligonucleotides of the probe, primers, reaction buffer, enzyme andother ingredients necessary for the amplification during the reaction,but is mixed with the main reaction mix after the completion of theamplification process, so that the melting profile can be measured. Thiscan be done by adding the second oligonucleotides to the main reactionvessel after the reaction completion. Alternatively, a single reactionvessel may have two separate chambers, one of which accommodates themain reaction mix; another chamber contains the second oligonucleotides.After the reaction completion, the liquids of two chambers are mixedtogether and melting profile is measured. In this way, it is notnecessary to open the reaction vessel, therefore reducing the risk ofcontamination.

In the methods of the present invention, at least one detectable labelmay be a fluorescent label, wherein step (b) further comprises the step(b1) obtaining cycle by cycle fluorescence emissions (FE) at variousmeasuring temperatures (MT), wherein said fluorescence emissions (FE) isa baseline corrected fluorescence (dR).

In any of the methods, said amplification may be an isothermalamplification or a thermal cycling amplification reaction comprising twoor more cycles of denaturing, annealing, and primer extension steps.

The consumption of probes may be achieved through hybridisation of theprobe or the one oligonucleotide of the probe, to the target sequence,which is followed by the incorporation of the probe or the oneoligonucleotide of the probe, into the amplified product, or whereinwhen the probe or the first oligonucleotide of the probe, can beincorporated into the amplified product, the probe or the firstoligonucleotide of the probe is extendable primer or is one of the pairof forward/reverse oligonucleotide primers.

The consumption of probes may be achieved through hybridisation of theprobe to the target sequence, which is followed by degradation of theprobe or the first and/or second oligonucleotide of the probe, whereinwhen the probe is degraded during the reaction, the reaction mixture maycomprise enzyme with nuclease activity.

In another aspect, the invention provides a kit for assaying for one ormore nucleic acid targets, which kit comprises a set of two or moreprobes comprising:

-   -   at least one probe in the set comprises a double-stranded        portion,    -   wherein each probe in the set comprises a detectable label or        detectable combination of labels which is/are capable of        producing a changeable signal which is characteristic for each        probe, and    -   wherein said two or more probes comprise the same detectable        label or different detectable labels with undistinguishable        emission spectra and wherein the melting characteristics of each        of such probes are different, each probe with double-stranded        portion has a signature melting temperature, whereas        single-stranded probes having no double stranded portion does        not have a signature melting temperature;    -   or at least one single-stranded probe which does not comprise a        double stranded portion, wherein said single-stranded probe is a        double dye labeled probe which causes detectable signal changes        upon hybridisation of the probe with a target nucleic acid        and/or degradation of the probe;    -   or at least two probes having double-stranded portions,        -   wherein a first probe has a melting temperature T_(m)1 in            terms of its double-stranded portion,        -   wherein a second probe has a melting temperature T_(m)2 in            terms of its double-stranded portion,    -   wherein T_(m)1>T_(m)2,        -   wherein the same labels are independently attached to the            first or/and second probes, wherein a reduction of any            melting peak at T_(m)1 and/or T_(m)2 provides indication of            consumption of the first and/or second probe(s)

In one aspect, the invention provides a method for assaying a sample forone or more target nucleic acids, said method comprising:

(a) contacting a sample comprising one or more target nucleic acids witha reaction mixture comprising:

-   -   a set of two or more probes, wherein at least one probe having        double-stranded portion may comprise    -   a first oligonucleotide which comprises a first region and        second region, wherein first region is substantially        complementary to part of one target nucleic acid, and at least        one second oligonucleotide which comprises a region which is        substantially complementary to the second region of the first        oligonucleotide, such that the first and second oligonucleotides        are capable of forming a double-stranded portion of the probe,    -   wherein each probe comprises a detectable label or detectable        combination of labels which is/are capable of producing a        changeable signal which is characteristic of the presence or        absence of a target nucleic acid, and    -   wherein at least two of the probes comprise the same detectable        label or different detectable labels with undistinguishable        emission spectra and wherein the melting characteristics        (melting temperature T_(m)) of each of such probes are        different, and are distinguishable in a melting profile        analysis;        (b) performing the reaction on the sample/reaction mixture,        wherein the reaction is a primer extension reaction under        extension conditions, wherein, when a target nucleic acid is        present, the probes act as primer and are extended, therefore        are consumed, wherein the first oligonucleotides of the        corresponding probe which are extendable primers, are hybridised        with target sequence, therefore are consumed during the primer        extension reaction, wherein the consumed oligonucleotides of        probes are no longer available to participate in forming double        stranded portion (the duplex) of the probe; and        (c) measuring, at least once, the melting profile of the        unconsumed probes in the reaction mixture by detecting the        signal(s) from the labels in those probes as a function of        temperature, wherein the melting profile provides an indication        of whether or not at least one target nucleic acid is present in        said sample.

In this embodiment, the first oligonucleotides of probes act as primers.In a primer extension reaction, a mixture of probes is added into thereaction mixture containing all ingredients for extension underextension conditions. If a particular target nucleic acid is present inthe reaction, the oligonucleotides of the corresponding probe arehybridised with target sequence, followed by extension and incorporationinto the primer extension product, therefore are consumed. The consumedoligonucleotides are no longer available to participate in forming thedouble stranded portion of the probe. In the melting profile analysis,the consumed probe can be seen as a peak reduced or missing.

In one embodiment of the present invention, the reaction mixture maycontain at least one probe which does not comprise the double-strandedportion. This kind of probe is referred to as single-stranded probe.Single-stranded probe may not show a distinguished melting profileduring measuring melting profile analysis, for example onesingle-stranded probe may not have a distinguished melting temperature(Tm). However, a single stranded probe may have a detectable signalchanges when hybridised to a target nucleic acid. During a meltingprofile analysis in the step (c), the consumed single stranded probe isdistinguishable from the rest of probes which comprise double strandedportion. For example, during a melting profile analysis in the step (c),if a detectable signal is increased while the melting profile of thereaction mixture does not reveal a significant melting curve change,i.e. the distinct melting signature for each probes with double-strandedportions are intact, it is can be inferred that the increased signal isdue to consumed single-stranded probe which does not have a signaturemelting profile.

In another aspect, the invention provides a method for assaying a samplefor one or more target nucleic acids, said method comprising:

(a) contacting a sample comprising one or more target nucleic acids witha hybridisation reaction mixture comprising:

-   -   a set of two or more probes, wherein at least one probe        comprises        -   a first oligonucleotide which comprises a first region which            is substantially complementary to part of one target nucleic            acid and a second region, and at least one second            oligonucleotide which comprises a region which is            substantially complementary to the second region of the            first oligonucleotide, such that the first and second            oligonucleotides are capable of forming a double-stranded            portion,    -   wherein said at least one probe comprises a detectable label or        detectable combination of labels which is/are capable of        producing a changeable signal which is characteristic of the        presence or absence of a double-stranded portion between the        first and second oligonucleotides of that probe, and    -   wherein at least two of the probes comprise the same detectable        label or different detectable labels with undistinguishable        emission spectra and wherein the melting characteristics of the        double-stranded portions between the first and second        oligonucleotides of each of such probes are different and are        distinguishable in a melting profile analysis;        (b) performing the hybridisation reaction on the sample/reaction        mixture under hybridisation conditions, wherein, when a target        nucleic acid is present, the first oligonucleotides of probes        which are substantially complementary to part of that target        nucleic acid are hybridised with target sequence, therefore are        consumed during the reaction, wherein the consumed        oligonucleotides of probes are no longer available to        participate in forming double stranded portion (the duplex) of        the probe; and        (c) measuring, at least once, the melting profile of unconsumed        probes in the reaction mixture by detecting the signal(s) from        the labels in those probes as a function of temperature, wherein        the melting profile provides an indication of whether or not at        least one target nucleic acid is present in said sample.

In one embodiment, the reaction mixture may contain at least one probewhich does not comprise a double-stranded portion. The probes or thefirst oligonucleotides of probes may act as hybridisation probe. In ahybridisation reaction, a mixture of probes is added into the reactionmixture containing all hybridisation ingredients under hybridisationconditions. If a particular target nucleic acid is present in thereaction, the oligonucleotides of the corresponding probe are hybridisedto the target sequence, therefore are consumed. The consumedoligonucleotides are no longer available to participate in forming thedouble stranded portion of the probe. In the melting profile analysis,the consumed probe can be seen as a peak reduced or missing.

In another aspect, the invention provides a method for assaying a samplefor one or more target nucleic acids, said method comprising:

(a) contacting a sample comprising one or more target nucleic acids withan amplification reaction mixture comprising:

-   -   (i) one or more pairs of forward/reverse oligonucleotide        primers, wherein the primer pairs are capable of amplifying one        or more target nucleic acids, if present in the sample,    -   (ii) a set of two or more probes, wherein at least one probe        comprises        -   a first oligonucleotide which comprises a first region which            is substantially complementary to part of one target nucleic            acid and a second region, and at least one second            oligonucleotide which comprises a region which is            substantially complementary to the second region of the            first oligonucleotide, such that the first and second            oligonucleotides are capable of forming a double-stranded            portion,    -   wherein said at least one probe comprises a detectable label or        detectable combination of labels which is/are capable of        producing a changeable signal which is characteristic of the        presence or absence of a double-stranded portion between the        first and second oligonucleotides of that probe, and    -   wherein at least two of the probes comprise the same detectable        label or different detectable labels with undistinguishable        emissions spectra and    -   wherein the melting characteristics of the double-stranded        portions between the first and second oligonucleotides of each        of such probes are different, and are distinguishable in a        melting profile analysis;        (b) performing an amplification reaction on the        sample/amplification reaction mixture wherein, when a target        nucleic acid is present, the first oligonucleotides which are        substantially complementary to part of that target nucleic acid        are hybridised with target sequence, therefore are consumed        during the amplification reaction;        (c) measuring, at least once, the melting profile of the        unconsumed probes by detecting the signal(s) from the labels in        those probes as a function of temperature,        wherein the melting profile provides an indication of whether or        not at least one target nucleic acid has been amplified in said        sample/amplification reaction mixture.

wherein a first probe of said at least two of the probes has a meltingtemperature T_(m)1 in terms of its double-stranded portion,

wherein a second probe of said at least two of the probes has a meltingtemperature T_(m)2 in terms of its double-stranded portion,

wherein T_(m)1>T_(m)2,

wherein the same labels are independently attached to the first andsecond probes,

wherein a reduction of any melting peak at T_(m)1 and/or T_(m)2 providesindication of consumption of the first and/or second probe(s).

Preferably, the above method includes step (d):

-   -   (i) comparing at least two melting profiles obtained in (c)        and/or    -   (ii) comparing a melting profile obtained in step (c)        -   with a previously-obtained melting profile of the same            probes or        -   with a melting profile of the same probes obtained in            parallel at the same time in control reactions, or        -   with a theoretical melting profile of the same probes            wherein a change in the melting profile provides an            indication of whether or not at least one target nucleic            acid has been amplified in said sample/amplification            reaction mixture.

In this embodiment, the reaction mixture comprises at least onesingle-stranded probe which does not have a signature melting profile.

The amplification reaction can be any amplification method, such as PCR,SDA, NASBA, LAMP, 3SR, ICAN, TMA, Helicase-dependent isothermal DNAamplification and the like. PCR is a preferred amplification method.

The amplification reaction mixture will comprise standard amplificationreagents. Amplification reagents can conveniently be classified intofour classes of components: (i) an aqueous buffer, often includingwithout limitation a magnesium salt, (ii) amplification substrates, suchas DNA or RNA, (iii) one or more oligonucleotide primers (normally twoprimers for each target sequence, the sequences defining the 5′ ends ofthe two complementary strands of the double-stranded target sequencewhen PCR is employed), and (iv) an amplification enzyme such as apolynucleotide polymerase (for example, Taq polymerase for PCR or RNApolymerase for TMA), or a ligase. Appropriate nucleoside triphosphateswill also generally be required. Additional reagents or additives canalso be included at the discretion of the skilled artisan and selectionof these reagents is within the skill of the ordinary artisan. Ofcourse, when the amplification reagents are used to cause both reversetranscription and amplification, then reverse transcription reagents arealso included in the amplification reagents. Selection of amplificationreagents, according to the method of amplification reaction used, iswithin the skill of the ordinary artisan.

In the methods described herein, a sample is provided which is suspectedto contain the target nucleic acid or the nucleotide variant ofinterest. The target nucleic acid contained in the sample may bedouble-stranded genomic DNA or cDNA if necessary, which is thendenatured, using any suitable denaturing method including physical,chemical, or enzymatic means that are known to those of skill in theart. A preferred physical means for strand separation involves heatingthe nucleic acid until it is completely (>99%) denatured. Typical heatdenaturation involves temperatures ranging from about 80° C. to about105° C., for times ranging from a few seconds to minutes. As analternative to denaturation, the target nucleic acid may exist in asingle-stranded form in the sample, such as single-stranded RNA or DNAviruses.

The denatured nucleic acid strands are then incubated witholigonucleotide primers and probes under hybridisation conditions, i.e.conditions that enable the binding of the primers or probes to thesingle nucleic acid strands. In some embodiments of the invention theannealed primers and/or probes are extended by a polymerizing agent.Template-dependent extension of the oligonucleotide primer(s) iscatalyzed by a polymerizing agent in the presence of adequate amounts ofthe four deoxyribonucleoside triphosphates (dATP, dGTP, dCTP, and dTTP),or analogues, in a reaction medium comprised of the appropriate salts,metal cations and pH buffering system. Suitable polymerizing agents areenzymes known to catalyze primer- and template-dependent DNA synthesis.The reaction conditions for catalyzing DNA synthesis with these DNApolymerases are well known in the art. Probes are consumed duringamplification.

An amplification primer can be a target-specific primer, which comprisesa 3′ priming portion which is complementary to a desired region oftarget nucleic acid. For SNP genotyping or detecting variantnucleotides, the amplification primer may be an allele-specific primer,wherein a terminal nucleotide of the primer is selected to be eithercomplementary to the suspected variant nucleotide or to thecorresponding normal nucleotide such that an extension product of theprimer is synthesised when the primer anneals to the diagnostic regioncontaining a particular nucleotide, but no such extension product issynthesised when the primer anneals to the diagnostic region containingno particular nucleotide of the target nucleic acid sequence.

Primer pairs of forward and reverse primers are included in theamplification reaction mixture such that, if a target nucleic acid ispresent in the sample, the primer pairs are capable of amplifying thattarget nucleic acid, preferably in an exponential manner.

In some embodiments, there will be 1-50, 1-25, 1-20 or 1-10 primer pairsin the reaction mixture. In other embodiments there will be 5-50, 5-25,5-20 or 5-10 primer pairs in the reaction mixture. As mentioned above,the forward or reverse primer in a particular primer pair might be auniversal primer which is common to more than one primer pair.

The amplification reaction mixture comprises a set of two or moreprobes. At least one probe comprises a double stranded portion. Theprobe may be a molecular beacon probe or the probe may comprise twostrands:

-   -   a first oligonucleotide which comprises a first region and a        second region, wherein the first region is substantially        complementary to part of one target nucleic acid, and    -   a second oligonucleotide which comprises a region which is        substantially complementary to the second region of the first        oligonucleotide,    -   such that the first and second oligonucleotides are capable of        forming a double-stranded portion of the probe.

The first oligonucleotide must be capable of binding, under appropriatehybridisation conditions, to part of at least one of the target nucleicacids. Preferably, each first oligonucleotide is specific to part ofonly one of the target nucleic acids. The first oligonucleotide willhave a first region whose nucleotide sequence is complementary orsubstantially complementary to the nucleotide sequence of part of one ofthe target nucleic acids. The length of this first complementary regionis preferably 6-100 nucleotides, more preferably 15-30 nucleotides.

The overall length of the first oligonucleotide is preferably 15-150nucleotides, more preferably 17 to 100 nucleotides, and most preferably20-80 nucleotides.

In some embodiments, where the reaction involves primer extension oramplification, the part of the target nucleic acid to which the firstoligonucleotide is complementary must fall within or overlap with thesequence to be amplified by the forward and reverse primers.Alternatively, the first oligonucleotide can be one of the amplificationprimers, for example, either the forward or reverse primer. In someembodiments, the first and/or second oligonucleotide is not a forward orreverse primer.

The second oligonucleotide comprises a region which is substantiallycomplementary to a second region of the first oligonucleotide. Thelength of this second region is preferably 4-100 nucleotides, morepreferably 15-30 nucleotides. The second region of the firstoligonucleotide may overlap or may not overlap with the first region ofthe first oligonucleotide.

The overall length of the second oligonucleotide is preferably 6-150nucleotides, more preferably 10 to 100 nucleotides, and most preferably12-80 nucleotides.

The first and second oligonucleotides may comprise 1-5 or 1-10 or morenucleotides that are not complementary to the target nucleic acid or tothe first oligonucleotide, respectively, at the 5′ or the 3′ end.

The second oligonucleotides of probes may not be present in the reactionmix during the reaction process. It is possible that during the PCRamplification, only the first oligonucleotides of probes are in thereaction mix. After the completion of amplification, the secondoligonucleotides of probes are added to reaction tube for meltingprofile analysis.

The oligonucleotide probe may comprise nucleotides, nucleotidederivatives, nucleotide analogs, and/or non-nucleotide chemicalmoieties. Modifications of the probe that may facilitate probe bindinginclude, but are not limited to, the incorporation of positively chargedor neutral phosphodiester linkages in the probe to decrease therepulsion of the polyanionic backbones of the probe and target (seeLetsinger et al., 1988, J. Amer. Chem. Soc. 110:4470); the incorporationof alkylated or halogenated bases, such as 5-bromouridine, in the probeto increase base stacking; the incorporation of ribonucleotides into theprobe to force the probe:target duplex into an “A” structure, which hasincreased base stacking; and the substitution of 2,6-diaminopurine(amino adenosine) for some or all of the adenosines in the probe; theincorporation of nucleotide derivatives such as LNA (locked nucleicacid), PNA (peptide nucleic acid) or the like.

Generally the 3′ terminus of the probe will be “blocked” to prohibitincorporation of the probe into a primer extension product. But in somepreferred embodiments of the present invention, some probes are alsoworking as primers and therefore are not blocked at the 3′ terminus.“Blocking” can be achieved by using non-complementary bases or by addinga chemical moiety such as biotin or a phosphate group to the 3′ hydroxylof the last nucleotide, which may, depending upon the selected moiety,serve a dual purpose by also acting as a label for subsequent detectionor capture of the nucleic acid attached to the label. Blocking can alsobe achieved by removing the 3′-OH or by using a nucleotide that lacks a3′-OH such as a dideoxynucleotide.

It will be readily understood that the term “probe” refers to aplurality of that type of probes, i.e. the reaction mixture does notcomprise merely a single molecule of that probe.

In some embodiments of the invention, the first region of said firstoligonucleotide does not overlap or does not substantially overlap withthe second region of said first oligonucleotide.

In other embodiments of the invention, the first region of the firstoligonucleotide is substantially overlapping with the second region ofsaid first oligonucleotide or the second region is embedded within thefirst region. In such embodiments, the T_(m) of the duplex of said firstoligonucleotide hybridised with the target sequence is preferably higherthan the T_(m) of the duplex of said first oligonucleotide hybridisedwith the second oligonucleotide such that if a target is present, thefirst oligonucleotide forms stronger hybrids with the target andconsequently melts at a higher temperature than the first/secondoligonucleotide duplex.

Preferably, said T_(m) of the duplex of said first oligonucleotidehybridised with the target sequence is at least 2 degrees or at least 5degrees higher than the T_(m) of the duplex of said firstoligonucleotide hybridised with the second oligonucleotide.

In other embodiments, the first oligonucleotide may comprise a thirdregion which is identical or substantially identical to the sequence ofa primer which is used in the amplification.

At least one probe of the present invention is capable of forming adouble-stranded portion. Because of this double-stranded portion, theprobe has a melting temperatures T_(m) and a signature melting profile.In particular, a mixture of multiple probes of the present inventionalso has a signature melting profile.

The melting temperature (T_(m)) is affected by a number of factors,including but not limited to, salt concentration, DNA concentration, andthe presence of denaturants, nucleic acid sequence, GC content, andlength. Typically, each probe of double stranded nucleic acids has aunique T_(m). At a temperature below a given T_(m) at least 50% ofnucleic acid duplex remains in duplex form. By contrast, at atemperature above a given T_(m), over 50% of nucleic acid duplexes areexpected to unwind into two single stranded oligonucleotide chains.

The T_(m) of any given DNA fragment can be determined by methods wellknown in the art. For example, one method in the art to determine aT_(m) of a DNA fragment is to use a thermostatic cell in an ultravioletspectrophotometer and to measure absorbance at 268 nm as the temperatureslowly rises. The absorbance versus temperature is plotted, presentingan S-shape curve with two plateaus. (See FIG. 1, for example). Theabsorbance reading half way between the two plateaus corresponds to theT_(m) of the fragment. Alternatively, the first negative derivative ofthe absorbance versus temperature is plotted, presenting a normaldistribution curve. The peak of the normal curve corresponds to theT_(m) of the fragment.

The T_(m) of a probe or T_(m)s of a mixture of multiple probes can alsobe determined by the nearest neighbour method and fine-tuned oraccurately determined in the presence of a double stranded DNA dye orlabels on the probe in a single reaction. For example, a reactionmixture containing a probe and appropriate buffer is heated from ahybridising temperature to the total denaturing temperature at a rate of0.01° C. to 3° C. per second. At the same time, the mixture isilluminated with light at a wavelength absorbed by the dye (label) andthe dye's (label's) emission is detected and recorded as an emissionreading. The first negative derivative of the emission reading withrespect to temperature is plotted against temperature to form a numberof normal curves, and each peak of the curve corresponds to the actualT_(m) of the probe. The curve is also know as “melting profile” or“hybridisation/dissociation profile”. The T_(m) or melting profile of aprobe can also be estimated by a computer program based on theory wellknown in the art.

For a multiplex detection, sets of multiple probes for multiple targetsequences are included in a reaction. In one embodiment, the differentprobes in a set of probes can comprise the same labels or labels withundistinguishable emission spectra. Each probe in such a set should havedifferent T_(m)s, therefore enabling the individual melting profiles tobe distinguished from one another. While the individual probe has amelting profile, the mixture of the multiple probes in the set also hasmelting profile which is characteristics for the set of the probes. Thereaction mixture may comprise one single stranded probe which does nothave a signature melting temperature.

In accordance with the present invention, multiple target nucleic acidsequences can be analysed in a single vessel by designing sets of probesthat hybridise to different target sequences and probes have differentmelting temperatures in terms of the probe's internal double-strandedportions. If a target sequence is present, its corresponding probe isconsumed. The sequence of the target can then be determined based on thecomparison of the melting profile of the probes before and after thereactions. Advantageously, the different probes in a set can be attachedwith the same label, allowing for monitoring at a single emissionwavelength. In one embodiment each probe in the set is attached with thesame labels, for example a fluorescent energy transfer pair or contactquenching pair, and more particularly, a first label which is afluorophore and a second label which is a quencher. On the other hand,the multiple sets of probes can be attached with different label pairsso that the sets of probes can be distinguished from one another basedon the distinguishable emission spectra.

In accordance with one embodiment, the method of analysing multipletargets uses a mixture of probes that are attached with different labelsthat have distinguishable emission spectra as well as probes that areattached with labels that have the same or overlapping emission spectra,but are distinguishable based on differences in melting temperatures ofthe internal double stranded portions of the probes.

In the case of probe having double stranded portion, the probe maycomprise two strands: a first oligonucleotide and a secondoligonucleotide, wherein said first oligonucleotide comprises a firstregion substantially complementary to a target nucleic acid (see forexample, FIG. 4). In one embodiment, the probe comprises a firstoligonucleotide and at least one second oligonucleotide (see forexample, FIG. 4A-J). The first oligonucleotide comprises a first regionwhich is substantially complementary to a target nucleic acid, andsecond region which is substantially complementary to a secondoligonucleotide(s) such that the first oligonucleotide and secondoligonucleotide(s) can bind together to form a double-stranded portion.The first region and second region can arranged in any order, such as 5′to 3′ or 3′ to 5′ (see for example FIG. 4A) or one embodied within oneanother (see for example FIG. 4B). It is preferred that if the firstoligonucleotide serves as primer, the first region and second region arearranged in an order as 3′ to 5′ (see for example FIG. 4A).

In one aspect, the first region of said first oligonucleotide is notoverlapping or not substantially overlapping with second region of saidfirst oligonucleotide (see for example FIG. 4A). In other words, thefirst region is complementary to a target sequence, while the secondregion may not be complementary to the target sequence. When the secondregion is not complementary to the target sequence, different probes mayhave the identical or substantially identical second region sequence andthe same second oligonucleotide may be shared between different probesin the set of probes. While the second oligonucleotides may be the sameamong the set of probes, the second regions of first oligonucleotides ofdifferent probes in the set may have the length and/or nucleotidesequence difference such that the T_(m) and the melting profile of theprobes are different.

In another aspect, the first region of said first oligonucleotide issubstantially overlapping with the second region of said firstoligonucleotide or the second region is embedded in the first region(see for example, FIG. 4B), wherein T_(m) of the duplex of said firstoligonucleotide hybridised with the target sequence is higher than theT_(m) of the duplex of said first oligonucleotide hybridised with thesecond oligonucleotide such that if a target is present, the targetforms stronger hybrids with the first oligonucleotide of the probe andconsequently the duplex of the target/first oligonucleotide melts at ahigher temperature than the duplex of second oligonucleotide/firstoligonucleotide. In this aspect, the first region may be longer than thesecond region or the second region may comprise mismatch nucleotideswhen hybridised with the second oligonucleotide. Binding of the firstoligonucleotide to the target nucleic acid prevents the secondoligonucleotide from binding to the first oligonucleotide of the probe.It is preferred that the T_(m) of the hybrid of said firstoligonucleotide and the target sequence is at least 2 degrees higherthan the T_(m) of the hybrid of said first oligonucleotide and thesecond oligonucleotide. It is more preferred that T_(m) of the hybrid ofsaid first oligonucleotide and the target sequence is at least 5 degreeshigher than the T_(m) of the hybrid of said first oligonucleotide andthe second oligonucleotide.

In yet another aspect, the first oligonucleotide comprises a thirdregion which is identical or substantially identical to a primersequence (see for example, FIGS. 4C and E). The third region may becomplementary or not complementary to the target sequence. Multipleprobes in a set of probes may comprise the same third region sequence.The primer identical to the third region sequence may act as a universalamplification primer. When the target specific probe (acting as primer)is running low at several cycles of amplification, the universal primercan take over and proceed to following cycles of amplification.

In some embodiments of the invention, the first and secondoligonucleotides are linked by a linker moiety. This linker moiety maycomprise nucleotides, nucleotide derivatives, nucleotide analogs or anon-nucleotide chemical linkage, i.e. the first and secondoligonucleotides might be a single stretch of contiguousoligonucleotides (FIG. 4K). In this embodiment, the probe can beunderstood as comprises a first oligonucleotide only (see for example,FIG. 4K), which comprises self-complementary regions capable of formingstem-loop structure, wherein said self-complementary regions aresubstantially complementary to each other which form the double-strandedportion of the probe. The stem part can be located any part ofoligonucleotide and has a length of 3 to 20 nucleotides. The 3′ part ofthe oligonucleotide is preferably complementary to the target sequence.It can have a blunt end, or 3′ protruding end or 5′ protruding end.Blunt end, or 3′ protruding end is preferred form.

The above described probe in which first and second oligonucleotides arelinked by a linker moiety can be regarded as molecular beacon probe oranalogue of molecular beacon probe (see U.S. Pat. Nos. 5,925,517 and6,10,3476, herein incorporated by reference). The reaction mixture inthe present invention may comprise one or more than one such molecularbeacon probes or its analogue. According to the claim 1 of U.S. Pat. No.5,925,517, a molecular beacon probe is a signaling unitary hybridizationprobe useful in an assay having conditions that include a detectiontemperature for detecting at least one nucleic acid strand containing apreselected nucleic acid target sequence, said probe comprising: asingle-stranded target complement sequence having from 10 to about 140nucleotides, having a 5′ terminus and a 3′ terminus, and beingcomplementary to the target sequence;

flanking the target complement sequence, a pair of oligonucleotide armsconsisting of a 5′ arm sequence covalently linked to said 5′ terminusand a 3′ arm sequence covalently linked to said 3′ terminus, said pairof oligonucleotide arms forming a stem duplex 3-25 nucleotides inlength, said stem duplex having a melting temperature above saiddetection temperature under said assay conditions; and

at least one interacting label pair, each pair comprising a first labelmoiety conjugated to the 5′ arm sequence and a second label moietyconjugated to the 3′ arm sequence,

said probe having, under said assay conditions in the absence of saidtarget sequence, a characteristic signal whose level is a function ofthe degree of interaction of said first and second label moieties, saidsignal having a first level at 10° C. below said melting temperature, asecond level at 10° C. above said melting temperature and a third levelat said detection temperature,

wherein under the assay conditions at the detection temperature and inthe presence of an excess of said target sequence, hybridization of thetarget complement sequence to the target sequence alters the level ofsaid characteristic signal from said third level toward the second levelby an amount of at least ten percent of the difference between the firstand second levels.

The analogue of molecular beacon probe in the present invention does notrequire that the stem duplex having a melting temperature above saiddetection temperature under assay conditions. The melting temperature ofstem duplex of the analogue of molecular beacon probe in the presentinvention is independence of the assay temperature, since we areinterested in measuring the melting profile of the probe. The meltingtemperatures of stem duplex of each probe are characteristic to eachprobe and distinguishable between probes. If the probe is consumedduring the reaction, its melting profile at the end of reaction willtell the difference. The 5′ arm or 3′ arm of molecular beacon probe orits analogue may be complementary to the target sequence or may bearbitrary sequences. The 5′ arm may be labeled with at least onefluorophore, the 3′ arm may be labeled with at least one quencher.

The single-stranded probe having no double-stranded portion, themolecular beacon probe, the analogue of molecular beacon probes or thefirst oligonucleotide of the probe with double-stranded portion iscapable of being consumed during amplification. Alternatively, both thefirst and second oligonucleotides of the probe with double-strandedportion are capable of being consumed during amplification. It ispreferred that the first oligonucleotide is designed to be consumed,while the second oligonucleotide may remain unchanged in a reaction.

The probe may be extendable, thereby acting as a primer. Alternatively,the first oligonucleotide is blocked at the 3′ end and secondoligonucleotide is blocked at the 3′ end, thereby being non-extendable.

Each probe comprises a detectable label which is capable of producing achangeable signal which is characteristic of the presence or absence ofdouble-stranded portion of that probe.

Furthermore, at least two of the probes comprise the same detectablelabel(s) or different detectable labels(s) with undistinguishableemission spectra.

The label on the probe may be a fluorophore, or the probe may comprisean interactive pair of labels, for example fluorophores and/ornon-fluorophore dyes. One example of such interactive labels is afluorophore-quencher pair. The label on the probe can be locatedanywhere as long as it interacts with other labels or other entitiessuch as G nucleotides.

In some embodiments, the first oligonucleotide comprises a first labeland the second oligonucleotide comprises second label. Preferably, thefirst label is a fluorophore and the second label is a quencher, or viceversa.

In other embodiments, the probe comprises two labels, the labels being aFRET pair. Preferably one label is on the first oligonucleotide and thesecond label is on the second oligonucleotide. Another preferred versionof probes is that the first oligonucleotide is dual labelled, the secondoligonucleotide is labelled or unlabelled.

Additionally, both the first and second oligonucleotides can alsocomprise a plurality of label moieties. For example, the firstoligonucleotide and/or the second oligonucleotide may comprise both afluorophore and a quencher.

Typically, the fluorophore and the quencher are attached to theoligonucleotides such that when the first oligonucleotide is bound to anunlabelled template sequence (e.g., a target), the fluorophore and thequencher are separated.

Alternatively, the fluorophore and the quencher are attached to theoligonucleotides such that when the first oligonucleotide is bound to anunlabelled template sequence (e.g., a target), the fluorophore and thequencher are brought into close proximity and hence the fluorophore isquenched.

“Fluorophore” as used herein to refer to a moiety that absorbs lightenergy at a defined excitation wavelength and emits light energy at adifferent defined wavelength.

Examples of fluorescence labels include, but are not limited to: AlexaFluor dyes (Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, AlexaFluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, AlexaFluor 660 and Alexa Fluor 680), AMCA, AMCA-S, BODIPY dyes (BODIPY FL,BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530/550, BODIPY 558/568,BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY650/665), Carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), Cascade Blue,Cascade Yellow, Cyanine dyes (Cy3, Cy5, Cy3.5, Cy5.5), Dansyl, Dapoxyl,Dialkylaminocoumarin, 4′,5′-Dichloro-2′,7′-dimethoxy-fluorescein,DM-NERF, Eosin, Erythrosin, Fluorescein, FAM, Hydroxycoumarin, IRDyes(IRD40, IRD 700, IRD 800), JOE, Lissamine rhodamine B, Marina Blue,Methoxycoumarin, Naphthofluorescein, Oregon Green 488, Oregon Green 500,Oregon Green 514, Pacific Blue, PyMPO, Pyrene, Rhodamine 6G, RhodamineGreen, Rhodamine Red, Rhodol Green,2′,4′,5′,7′-Tetra-bromosulfone-fluorescein, Tetramethyl-rhodamine (TMR),Carboxytetramethylrhodamine (TAMRA), Texas Red and Texas Red-X.

As used herein, the term “quencher” includes any moiety that is capableof absorbing the energy of an excited fluorescent label when it islocated in close proximity to the fluorescent label and capable ofdissipating that energy. A quencher can be a fluorescent quencher or anon-fluorescent quencher, which is also referred to as a dark quencher.The fluorophores listed above can play a quencher role if brought intoproximity to another fluorophore, wherein either FRET quenching orcontact quenching can occur. It is preferred that a dark quencher whichdoes not emit any visible light is used. Examples of dark quenchersinclude, but are not limited to, DABCYL(4-(4′-dimethylaminophenylazo)benzoic acid) succinimidyl ester,diarylrhodamine carboxylic acid, succinimidyl ester (QSY-7), and4′,5′-dinitrofluorescein carboxylic acid, succinimidyl ester (QSY-33),quencherl, or “Black hole quenchers” (BHQ-1, BHQ-2 and BHQ-3),nucleotide analogs, nucleotide G residues, nanoparticles, and goldparticles.

The interactive label pair can form either FRET or a contact quenchingrelationship. The quencher is preferably a non-fluorescent entity. Thequencher may be a nanoparticle. A nanoparticle may be a goldnanoparticle. It is also possible that the quencher is a G residue ormultiple G residues.

The label or combination of labels on each probe are capable ofproducing a changeable signal which is characteristic of each probe.

At least one label is attached to the probe or the first oligonucleotideor to the second oligonucleotide of the probe. The label eitherincreases or decreases fluorescence emission when the firstoligonucleotide is bound to the second oligonucleotide.

Preferably, the probe comprises a first label and a second label,wherein at least one label is capable of producing a detectable signaland wherein the signal strength is affected by the proximity of the twolabels.

In some embodiments of the invention, the first label is attached to onestrand of the double stranded portion and the second label is attachedto the opposite strand of the double stranded portion of the probe suchthat said first label and second label are in close proximity when theprobe's internal duplex is formed.

In other embodiments, the first label is attached to the firstoligonucleotide, and the second label is attached to the secondoligonucleotide such that said first label and second label are in closeproximity when the probe's internal double-stranded portion is formed.

In some embodiments of the invention, the first oligonucleotide does notcomprise a label. In other embodiments, the second oligonucleotidecomprises a single label which is capable of changing fluorescenceemission when hybridised with the first oligonucleotide.

In some embodiments, the first label is attached to the firstoligonucleotide and the second label is attached to the secondoligonucleotide such that said first label and second label are in closeproximity when the probe's internal duplex is formed. Preferably, thefirst label is attached to the second region of the firstoligonucleotide and the second label is attached to the region of thesecond oligonucleotide which is complementary to the second region ofthe first oligonucleotide such that the first and second labels arebrought into close proximity upon formation of the probe's internalduplex. Examples of such embodiments are shown in FIGS. 4A, 4B and 4C.

In some aspects of the invention, the first oligonucleotide of the probedoes not comprise a label, but the second oligonucleotide of the probecomprises at least one, preferably two, labels.

In one embodiment of this aspect, the second oligonucleotide comprises afirst label and a second label. The first label is attached at or nearone end of second oligonucleotide and the second label is attached at ornear the other end of the second oligonucleotide, whereby when thesecond oligonucleotide is not hybridised with the first oligonucleotide,the second oligonucleotide is in a random-coiled or a stem-loopstructure which brings the first label and second label in closeproximity. When the second oligonucleotide is hybridised with the firstoligonucleotide, the two labels are held away from each other. Examplesof such embodiments are shown in FIGS. 4D, 4E and 4F.

As is known in the prior art, a double-labelled oligonucleotide can forma random-coiled structure when it is in single-stranded form and atcertain permissive temperatures. This kind of linear oligonucleotideprobes in solution behaves like a random coil: its two ends occasionallycome close to one another, resulting in a measurable change in energytransfer. However, when the probe binds to its template, theprobe-template hybrid forces the two ends of the probe apart, disruptingthe interaction between the two terminal moieties, and thus causing afluorescence emission change. In the present invention, this kind of adouble-labelled oligonucleotide may be included in the set of two ormore probes. The feature of probe having not obvious melting temperatureis distinguishable from rest of probes having double stranded portionand signature melting temperatures.

A double-labelled oligonucleotide can also form a stem-loop structureknown as molecular beacon. Molecular beacon probes are single-strandedoligonucleic acid probes that can form a hairpin structure in which afluorophore and a quencher are usually placed on the opposite ends ofthe oligonucleotide. At either end of the probe short complementarysequences allow for the formation of an intramolecular stem, whichenables the fluorophore and the quencher to come into close proximity.The loop portion of the molecular beacon is complementary to a targetnucleic acid of interest. Binding of this probe to its target nucleicacid of interest forms a hybrid that forces the stem apart. This causesa conformation change that moves the fluorophore and the quencher awayfrom each other and leads to a more intense fluorescent signal (Tyagi S,and Kramer F. R., Nature Biotechnology, Vol. 14, pages 303-308 (1996);Tyagi et al., Nature Biotechnology, Vol. 16, pages 49-53 (1998); Piateket al., Nature Biotechnology, Vol. 16, pages 359-363 (1998); Marras S.et al., Genetic Analysis: Biomolecular Engineering, Vol. 14, pages151-156 (1999); Tpp I. et al, BioTechniques, Vol 28, pages 732-738(2000)).

In the present invention, molecular beacon probe or its analogue probemay be included in the probe set in a reaction, where each molecularbeacon probe has a signature melting temperature therefore isdistinguishable during melting profile analysis.

In another embodiment, one type of the second oligonucleotides of thelinear probe with double-stranded portion, which can be a molecularbeacon-like oligonucleotide, is part of a double-stranded portion of aprobe. The differences of this kind of probe from other probes are thatthe second oligonucleotide may not hybridise to the target sequence, butto the second region of the first oligonucleotide which may be unrelatedto the target sequence. The second oligonucleotide may be capable ofhybridising to the target sequence, but it is designed that it may notbe able to hybridise to the target sequence in a real amplificationreaction. The second oligonucleotide may contain sequence which isincapable of binding a target sequence strongly. During an extension andannealing step of amplification, the temperature may be too high for thesecond oligonucleotide to hybridise to the target sequence. While duringthe signal collection step, which often is carried out after anextension step, the temperature may be low, but the target sequence maybe unavailable for the second oligonucleotide to hybridise, as thetarget sequence to be amplified may become double-stranded due to theextension of amplification primer already take place. Therefore, duringsignal collection step, the second oligonucleotide may only be able tohybridise to the unconsumed first oligonucleotide of the probe.

In another aspect, the first oligonucleotide does not comprise a labelwhereas the second oligonucleotide comprises a label. When the secondoligonucleotide hybridises to the first oligonucleotide to form thedouble-stranded portion of the probe, the label changes its detectablesignal emission relative to the emission of the label in thesingle-stranded form of the second oligonucleotide. This may be becauseeither the label is brought into close proximity with a nucleotide ornucleotides in the first oligonucleotide, or the label is held away witha nucleotide or nucleotides in the second oligonucleotide.

It is known in the prior art that the emission of a fluorescence dye canbe changed when in close proximity to certain nucleotides, for example aG nucleotide.

In other embodiments, the first oligonucleotide of the probe does notcomprises a label, and the probe comprises two second oligonucleotideswhich are capable of hybridising adjacently or substantially adjacentlyto different parts of the second region of the first oligonucleotide,wherein one of the second oligonucleotides is attached with a firstlabel, and the other second oligonucleotide is attached with a secondlabel, such that when the two second oligonucleotides are hybridised tothe first oligonucleotide, the two labels are brought in close proximityand one label affects the signal from the other.

This close proximity may, for example, cause either FRET or contactquenching relationship. The two second oligonucleotides that hybridiseto the first oligonucleotide are part of the probe. The two labelledsecond oligonucleotides are designed to not hybridise to amplifiedtarget sequence, but to hybridise to the unconsumed firstoligonucleotide. Examples of such embodiments are given in FIGS. 4I and4J.

In yet other embodiments, the first and second oligonucleotides of aprobe are joined by a linker moiety which comprises nucleotides or anon-nucleotide chemical linker, allowing the first oligonucleotide andsecond oligonucleotide to form a stem-loop structure, wherein the firstand second oligonucleotides are each labelled such that, when the probeforms an internal stem-loop structure, the labels are brought into closeproximity and one label affects the signal from the other.

The linker may, for example, be a simple moiety of formula (CH₂)_(n) ora linker which is functionally equivalent thereto. (n is preferably1-100 or 1-50). Preferably, the linker is an oligonucleotide which iscontiguous with the first and second oligonucleotides. The loop ispreferably complementary to the target sequence.

At least two of the probes in the amplification reaction mixturecomprise the same detectable label or a different detectable label withundistinguishable emission spectra.

In multiplex reactions, two or more probes are used to assay for thepresence of two or more target nucleic acids. This does not necessarymean, however, that different distinguishable labels are needed for eachof the different probes. Each probe will have a characteristic meltingprofile which will be dependent on the features of its internaldouble-stranded portion. Therefore, provided that two or more probeshave distinguishable melting characteristics, the same label(s) can beused for those probes. In other words, different probes which arelabelled with the same labels or labels having undistinguishableemission spectra must have different melting characteristics, preferablydifferent melting temperatures (T_(m)). These different meltingcharacteristics will allow the individual probes to be identified and/orquantified in step (c).

As used herein, the term “melting characteristics” includes the meltingprofile of a probe (preferably measured by detecting the signal from thelabel(s) on that probe as function of temperature) and/or the meltingtemperature (T_(m)) of a probe.

When the melting characteristics of probes are said to be “different”,it will be understood that the differences are to be measured under thesame or control conditions.

In some embodiments of the invention, two or more probes are labelledwith the same detectable label. For example, at least 2, 3, 4, 5, 6, 7,8, 9, 10, 20 or 30 or more different probes may all be labelled with thesame label.

In step (b), an amplification reaction is performed on thesample/amplification (or sample/hybridisation) reaction mixture wherein,when a target nucleic acid is present, the probe which are substantiallycomplementary to part of that target nucleic acid are consumed duringthe amplification reaction.

The amplification reaction can be carried out under conditions known inthe art such that the probes which are substantially complementary topart of the target nucleic acid are consumed.

Preferably, the amplification comprises at least one denaturing step, atleast one annealing step and at least one primer extension step.

More preferably, the amplification is a thermal cycling amplificationcomprising two or more denaturing, annealing, and primer extensionsteps.

Preferred amplification reactions include PCR, SDA, NASBA, LAMP, 3SR,ICAN, TMA, Helicase-dependent isothermal DNA amplification and the like.PCR is a preferred amplification method. If the amplification is PCR,the conditions comprise thermally cycling the reaction.

When a target nucleic is present in the sample, at least some of theprobes which are complementary to part of the target nucleic acids willhybridise thereto under appropriate reaction conditions. The probes willtherefore be consumed.

The consumption of the probes is achieved through hybridisation of theprobe to the target sequence, which may be followed by incorporation ofthe probe into the amplified product or/and degradation of the probeduring the amplification step. In other words, after consumption, theprobe is no longer able to reconstitute the probe of which it previouslyformed a part.

In an amplification reaction, in the case of probe having two strands,the probe of the present invention can be constituted by adding thefirst and second oligonucleotides in the reaction at any ratio of thesecond oligonucleotide to the first oligonucleotide, for examplepreferably more than 1, or may be more than 0.1 and less than 1. Thusthe first and second oligonucleotides may be added to the amplificationreaction mixture independently.

Depending on the type of assay and the type of label that is actuallyused, the signal from the probe (e.g. fluorescence emission) may eitherincrease or decrease when the probe or the first oligonucleotide of theprobe is consumed. Reference is made, for example, to the embodimentsshown in FIG. 4. In FIGS. 4A-4C, the consumption of the probe leads toan increase in fluorescence because the first oligonucleotide attachedwith fluorophore is consumed, thus allowing the fluorophore to emit itssignal. On the contrary, in FIGS. 4D-4F, the consumption of the firstoligonucleotide releases the dual end-labelled second oligonucleotideallowing the fluorophore and quencher to juxtapose one another, thusleading to a reduction in signal from the fluorophore.

In one embodiment, the probe is extendable and acts as the forward orreverse primer. The first oligonucleotide of the probe may be one of theamplification primers, which is capable of being incorporated into anamplified product, thereby being consumed (see for example FIG. 6). In aPCR, the first oligonucleotide of the probe pairs with anotheramplification primer for the opposite strand to perform theamplification. During signal collection step, or the step of measuringthe melting profile of the probe, the incorporated first oligonucleotideof the probe is not available to form the duplex of the probe's internaldouble stranded portion. The amount of unconsumed first oligonucleotideof the probe which is able to form the probe duplex can be measured anddetermined, and the signal can be transformed to the amount of the firstoligonucleotide being consumed, thereby determining which or how much ofthe target sequence is present in a sample. When the firstoligonucleotide acts as a primer, the second oligonucleotide preferablydoes not act as a primer.

In another embodiment of the present invention, the single-strandedprobe or the first oligonucleotide of the probe having two strands isdegraded during amplification. The first oligonucleotide may, forexample, comprise nucleotides or non-nucleotide chemicals which aresensitive to a digestion agent. In a further example, upon hybridisingto the target sequence the probe or the first oligonucleotide can bedegraded by the digestion agent. For example, the probe or the firstoligonucleotide can comprise RNA nucleotides. When the firstoligonucleotide hybridises to the target sequence, it can be degraded byenzymes with the RNase H activity. It can be designed such that thefirst oligonucleotide is not able to be degraded when hybridised to thesecond oligonucleotide to form the double stranded portion of the probe.The first oligonucleotide can also be degraded by an exonuclease. Forexample, the 3′ nucleotide of the first oligonucleotide can be degradedby the 3′ exonuclease activity of a polymerase. The firstoligonucleotide can also be degraded by an endonuclease, for example bya restriction enzyme upon hybridisation to a target nucleic acid.

It is preferred that the single-stranded probe or the firstoligonucleotide of the probe having two strands is degraded by the 5′exonuclease activity of a DNA polymerase, such as Taq DNA polymerase. Inthis embodiment, the first oligonucleotide of the probe may be blockedat the 3′ end, and hence is therefore non-extendable. The firstoligonucleotide hybridises to the target sequence in the region boundedby the forward and reverse primers and can be degraded by a nucleaseactivity, such as 5′ exonuclease activity of Taq polymerase during PCRamplification (see for example FIG. 8). Alternatively, the firstoligonucleotide of the probe is not blocked at the 3′ end, and hence istherefore extendable. The first oligonucleotide hybridises to the targetsequence and can be extended by a polymerase. An amplification primerupstream of the first oligonucleotide is also extended. When theextension of the amplification primer encounter the extension strand ofthe first oligonucleotide, the entire extension strand of the firstoligonucleotide can be degraded by a nuclease activity, such as 5′exonuclease activity of Taq polymerase during PCR amplification (see forexample, FIG. 7).

In yet another embodiment of the present invention, the consumption ofthe probe can simply be the hybridisation of the probe to the targetsequence, whereby the hybridised probe is not available to form doublestranded duplex of the probe (see, for example, FIG. 5). It is preferredthat amplification is designed to produce a single-stranded product sothat the first oligonucleotide of the probe can hybridise to the targetsequence during the annealing step and/or after the extension step. Themethods to produce single-stranded products can be asymmetric PCR, or amethod described in PCDR (PCT/GB2007/003793). The single-strandedamplification strand can form a strong hybrid with the firstoligonucleotide; therefore the first oligonucleotide can be regarded asconsumed, as it is not readily available to form the internal probehybrid.

In yet another embodiment of the present invention, the firstoligonucleotide of the probe may play a role as a nested inneramplification primer. The first oligonucleotide and an outeramplification primer anneal to the same strand of the target nucleicacid. The outer amplification primer and the first oligonucleotide maybe both extended upon hybridisation to the target nucleic acid. Theextension strand of the first oligonucleotide may be displaced duringthe extension of the outer primer if the DNA polymerase comprises adisplacement activity. The extension strand of the first oligonucleotidemay be degraded during the extension of the outer primer if the DNApolymerase comprises a 5′ exonuclease activity.

In some embodiments, an amplification condition can be designed suchthat at some stages of the amplification, even when the target ispresent, the probe is not consumed, whereas at other stages of theamplification the probe is consumed. For example, if the amplificationis PCR, at the some thermal cycles of amplification, the probe may notbe consumed, because the annealing and extension temperature are set toohigh for the probe to bind. The amplification thermal condition can bedesigned such that the probes can be consumed at some stages, or thelast cycle of the amplification. For example, after thermal cycling thePCR vessels are incubated at a temperature which is set lower thaneither annealing and extension temperatures of the thermal cycling. Thislow temperature allows the probe to hybridise to the target nucleicacids, resulting in either extension or degradation of the hybridisedprobe.

Step (b) may further comprise the step (b1) of measuring cycle by cyclefluorescence emissions (FEs) at various measuring temperatures (MTs).The measuring temperatures (MTs) refers to the temperature at which anemission reading of the label in the probe is taken cycle by cycle todetermine the emission amount of a probe.

The emission of a label on the probe is preferably obtained, detectedand/or recorded in each cycle in a reaction after the reaction mixtureis illuminated or excited by light with a wavelength absorbed by thelabel. The term “cycle by cycle” refers to measurement in each cycle.The emission reading at a measuring temperature is taken to calculatethe emission amount of a remaining probe in a cycle. Emission can bedetected, recorded, or obtained continuously or intermittently.

In a continuous recording process, the emission of the probe ismonitored and recorded, for example, every 50 ms, every 100 ms, every200 ms or every 1 s, in each cycle of, for example, a PCR reaction. Athree dimensional plot of time, temperature and emission can be formed.In any given cycle, the emission reading at a time point thatcorresponds to a desired MT is taken to determine the emission amount ofthe probe in the cycle. In an intermittent recording process, theemission reading is taken only when the reaction temperature reaches adesired MT in each cycle.

For the probes which are consumed by incorporating into the amplifiedproduct or being degraded by a digestion agent, the obtaining of thecycle by cycle fluorescence emissions (FE) is preferably performed afterthe completion of the extension step at each cycle. For the probes whichare consumed by being hybridised to the target sequence, the obtainingof the cycle by cycle fluorescence emissions (FE) may be performedbefore the completion of the extension step at each cycle.

Fluorescence emissions (FE) is used herein to refer to baselinecorrected fluorescence (dR). Normally, for each well (reaction) and eachoptical path the raw fluorescence data are fitted over the specifiedrange of cycles using a linear least mean squares algorithm (or othersuch algorithm) to produce a baseline. The value of the baselinefunction is calculated for every cycle and subtracted from the rawfluorescence to produce the baseline corrected fluorescence (dR).

The fluorescence intensity data (amplification plots) can be describedas a two-component function: a linear component or background and anexponential component that contains the relevant information. To isolatethe exponential component, the linear contribution to the fluorescencecan be estimated and subtracted. It is a three-step process that iscarried out for each amplification plot (i.e. each reaction and eachlabel):

-   -   1. Identify the range of cycles during which all contributions        to the fluorescence are strictly linear (no exponential increase        in fluorescence).    -   2. Using the fluorescence intensity values during the cycles        determined above, fit the data to a straight line (a function        predicting the contribution of the linear components throughout        the reaction).    -   3. Subtract the predicted background fluorescence intensity        during each cycle. The resulting curve corresponds to the change        in fluorescence due to DNA amplification.

When the amplification reaction mixture comprises “n” probes formultiplex detection of “n” nucleic acid targets, first probe has amelting temperature of T_(m)1, the second probe has a meltingtemperature of T_(m)2, the third probe has a melting temperature ofT_(m)3, the k-th probe has a melting temperature T_(m)k, and the n-thprobe has a melting temperature of T_(m)n, wherein T_(m)1>T_(m)2>T_(m)3>. . . T_(m)k . . . >T_(m)n, wherein n and k are positive integers,1≦k≦n, and n≧2.

The percentage of a probe's double-stranded form out of the total amountof probes at a particular temperature or at series temperatures may bedetermined experimentally or predicted which can be done by a computerprogram. Since the first negative derivative of a probe's meltingemission with respect to temperature is plotted to form a normaldistribution curve, an ordinary person skilled in the field ofstatistics could readily define a MT at which a percentage of the totalnumber of a given probe is in duplex form or in single-stranded (i.e.separated) form. Accordingly, a measuring temperature is a temperatureat which no more than 20%, for example, of a probe is in single-strandedform. A table may be created listing percentages of double-stranded (ds)and single-stranded forms(s) of each probe at each temperature.

A first fluorescence emission FEa can be obtained at a measuringtemperature MTa, at which more than 50% of first probe is in duplexform; second fluorescence emission FEb can be obtained at a measuringtemperature MTb, at which more than 50% of second probe is in duplexform; k-th fluorescence emission FEk can be obtained at a measuringtemperature MTk, at which more than 50% of k-th probe is in duplex form;n−1 fluorescence emissions FE(n−1) can be obtained at a measuringtemperature MT(n−1), at which more than 50% of (n−1)-th probe is induplex form, n-th fluorescence emission FEn can be obtained at ameasuring temperature MTn, at which more than 50% of n-th probe is induplex form, and optionally a fluorescence emission FE0 can be obtainedat a measuring temperature MT0, at which no more than 10% of first probeis in duplex form.

Although the above-mentioned 50% is a preferred amount of probe in theduplex form, it should be appreciated that any percentage can be used,such as 40%, 55%, 70% or 80%. It is preferred that in the step obtainingcycle by cycle a fluorescence emission FEk at a measuring temperatureMTk, at which no more than 30% of (k−1)-th probe is in the probe'sinternal duplex form. It is even preferred that in the step obtainingcycle by cycle a fluorescence emission FEk at a measuring temperatureMTk, at which no more than 20% of (k−1)-th probe is in the probe'sinternal duplex form.

The step (b) may further comprise the step (b2) determining cycle bycycle Actual Consumed Amount of fluorescence emission from consumedprobe for each probe, wherein the Actual Consumed Amount of fluorescenceemission t of k-th probe is depicted as ACA_(k). At a particularmeasuring temperature (MTa), where a probe has certain percentage (dska)% in ds (double-strand) form, the fluorescence emission FE at thismeasuring temperature MTa contributed by first probe will be (ds1a)%*(ACA₁), contributed by the second probe will be (ds2a) %*(ACA₂),contributed by the k-th probe will be (dska) %*(ACA_(k)). For example,at 60° C. 70% of probe 1 is in ds form; at 50° C. 80% in ds. The FEcontributed by the probe 1 at 60° C. will be 70%*(ACA₁); FE contributedby the probe 1 at 50° C. will be 80%*(ACA₁). If multiple probes arepresent, the FE will be the total amount contributed by consumed probesof all probes. The calculation of Actual Consumed Amount (ACA) can usethe following formula:

At temperature a, the total fluorescence emission will be

FEa=(ACA1)*(ds1a)%+(ACA2)*(ds2a)%+(ACA3)*(ds3a)% . . . +(ACAn)*(dsna)%

At temperature b, the total fluorescence emission will be

FEb=(ACA1)*(ds1b)%+(ACA2)*(ds2b)%+(ACA3)*(ds3b)% . . . +(ACAn)*(dsna)%

At temperature c, the total fluorescence emission will be

FEc=(ACA1)*(ds1c)%+(ACA2)*(ds2c)%+(ACA3)*(ds3c)% . . . +(ACAn)*(dsna)%

And so on. The individual ACA can be calculated from the above formulas.“*” denotes “multiply by”.

It is preferred that the Actual Consumed Amount of each probe isobtained though a computer program which performs the above calculation.Alternatively, the calculation can be done manually.

For example, in an amplification reaction, there are three probes forthree target sequences. At temperature 65° C., 5% of the first probe isin duplex form, 0% of the second probe and 0% of third probe is induplex form. At 60° C., 60% of the first probe is in duplex form, 5% ofsecond probe is in duplex form, 0% of the third probe is in duplex form.At 55° C., 90% of the first probe is in duplex form, 55% of second probeis in duplex form, 5% of third probe is in duplex form. At 45° C., morethan 95% of all probes are in duplex form. The first fluorescenceemission is collected at 60° C., which is FE60, the second fluorescenceemission is collected at 55° C., which is FE55, the third fluorescenceemission is collected at 45° C., which is FE45. Optionally before thefirst fluorescence emission is collected, a fluorescence emission iscollected at 65° C. or above, which is FE65. The FE contributed by theactual consumed amount ACA from individual probe is calculated as ds%*(ACA). See the table below:

probe 1 probe 2 probe 3 fluorescence ds % ACA1 ds % ACA2 ds % ACA3emission 65° C. 5  5% ACA1 0  0% ACA2 0 0% ACA3 FE65 60° C. 60 60% ACA15  5% ACA2 0 0% ACA3 FE60 55° C. 90 90% ACA1 55 55% ACA2 5 5% ACA3 FE5545° C. 95 95% ACA1 95 95% ACA2 95 95% ACA3  FE45 FE65 = 5% * ACA1 + 0% *ACA2 + 0% * ACA3 ≈ 0 FE60 = 60% * ACA1 + 5% * ACA2 + 0% * ACA3 (1) FE55= 90% * ACA1 + 55% * ACA2 + 5% * ACA3 (2) FE45 = 95% * ACA1 + 95% *ACA2 + 95% * ACA3 (3)

The individual ACA can be calculated from the above formulas. If weassume 5%*ACA can be neglected. The approximate ACA can be calculatedfrom (1), (2) and (3), where

ACA1=(EF60)/0.6

ACA2=((EF55)−0.9/0.6*(EF60))/0.55

ACA3=(EF45)/0.95−(EF60)/0.6−(EF55−(0.9/0.6)*(EF60))/0.55

As a PCR mixture undergoes thermal cycling, the fluorescence emissionand actual consumed amount (ACA) are recorded (calculated) and plottedover the number of cycles to form an emission versus cycle plot. In theinitial cycles, there is little change in the emission amount whichappears as a baseline or a plateau in the plot. As thermal cyclingcontinues, an increase in emission amount above the baseline may beexpected to be observed, which indicates that the amplified product (orconsumed probes) has accumulated to the extent that fluorescenceemission of a probe in the presence of the amplified product (orconsumed probe) exceeds the detection threshold of a PCR instrument. Anexponential increase in emission amount initiates the exponential phaseand eventually reaches another plateau when one of the components in thePCR mixture becomes limiting. The plot usually produces an S-shape curvewith two plateaus at both ends and an exponential phase in the middle.In the exponential phase, the emission amount of the probe is increasingto (1+E) fold over the previous amount of each cycle, wherein E is theefficiency of amplification, which ideally should be 100% or 1. It iscommonly known that the higher the starting amount of the nucleic acidtemplate from which a product is amplified, the earlier an increase overbaseline is observed. As is well known in the art, the emission versuscycle plot provides significant information for attaining the initialcopy number or amount of the nucleic acid template.

As is known in the field of real-time PCR, the unknown amount of anucleic acid template may be quantified by comparing the emission versuscycle plot of the template with standardized plots.

In one embodiment of the present invention, when a plurality of nucleicacid templates are amplified to form a plurality of amplified products,each product is preferably compared with a standard curve formed by thesame product. A single product per dilution per PCR mixture can be usedto form the standard curve. Preferably, at each dilution, a plurality ofproducts are placed in a single PCR mixture and emission readings ofeach probe can be measured and plotted to form a standard curve based onmethods described in the present invention.

The starting amount of a nucleic acid template in a sample can also bedetermined by normalizing the template to a house-keeping gene or anormalizer in relative relationship to a calibrator without using astandard curve.

In one embodiment of the present invention, a plurality of nucleic acidtemplates of interest are amplified and quantified in a single PCRmixture. The starting amount of each nucleic acid template cansimultaneously be calculated and normalized to a normalizer. It is alsocontemplated that a plurality of nucleic acid templates and a normalizertemplate can be monitored and amplified in the same PCR reaction. It isfurther contemplated that more than one housekeeping template ornormalizer can be amplified along with multiple nucleic acid templatesin a single PCR reaction. It is further contemplated that the relativeamount among these templates or the ratios between or among thesetemplates can be determined from a single PCR mixture.

Step (c) comprises measuring, at least once, the melting profile of thedouble-stranded portions between the first and second oligonucleotidesof unconsumed probes by detecting the signals from the labels in thoseprobes as a function of temperature, wherein the melting profileprovides an indication of whether or not at least one target nucleicacid has been amplified in said sample/amplification reaction mixture.This step can be performed immediately after PCR amplification if thereaction mix contains the second oligonucleotides of probes, or is doneby adding the second oligonucleotides of probes, if which are notpresent in the reaction, into the main reaction mix, then performing themelting curve analysis.

To determine the melting (profile) curve for each probe or each set ofprobes, the reaction mixture is illuminated with light that is absorbedby the labels of the probes and the fluorescence of the reaction ismonitored as a function of temperature. More particularly, thefluorescence of the labels is measured as the temperature of the sampleis raised (or decreased) until a baseline level of fluorescence isachieved.

The data may be presented as fluorescence vs. temperature plots or asfirst derivative plots of fluorescence vs. temperature, for example. Thetwo plots are interchangeable, but each focuses the viewer's attentionon different aspects of the data. The melting peak (or T_(m)) is bestviewed on derivative plots. However, the broadening of the transitionand appearance of low melting transitions are easier to observe onfluorescence vs. temperature plots. The point at which there is a shiftin the rate of decrease or increase of fluorescence can be more easilyidentified by viewing a plot of the first derivative of the fluorescencevs. temperature. The point of maximum rate of change is considered themelting temperature of the probe duplex. If one probe has a higherT_(m), it forms a stronger probe duplex and will consequently melt at ahigher temperature than another probe. The distinctly different meltingtemperatures of different probes allow identification of which probe isconsumed during amplification (if the probes have the same label).

In some methods of this invention, fluorescence is monitored as afunction of a denaturing gradient. Independent from the type ofgradient, however, what is actually monitored is the change influorescence caused by the dissociation of the two strands of thedouble-stranded portion of the probe. The denaturing gradient may be athermal gradient. In other words, the invention is illustrativelydirected to a method, characterized in that during or subsequent to the(preferably PCR) amplification, temperature dependent fluorescence ismonitored. It is often desirable, however, if the monitoring oftemperature-dependent fluorescence is part of a homogeneous assay formatsuch that (PCR) amplification and monitoring temperature dependentfluorescence are carried out in the same reaction vessel withoutintermediate opening of the reaction chamber.

Melting profile analysis may be obtained by monitoringtemperature-dependent fluorescence during melting or hybridisation.Usually, melting curve analyses are performed as slowly as possible inorder to generate precise and highly reproducible data, in order toobtain an exact determination of the melting point, which is defined asthe maximum of the first derivative of a temperature versus fluorescenceplot. However, if the selected time parameters are comparatively short,certain advantages may be seen.

A melting profile may be measured after completion of amplification(post-amplification melting profile), and/or may be measured duringamplification at each cycle or at selected cycles (mid-amplificationmelting profile).

In one embodiment, the temperature-dependent fluorescence is monitoredafter completion of a PCR reaction. In an alternative embodiment, thetemperature-dependent fluorescence is monitored in real-time during aPCR reaction.

In one embodiment during said measuring, the amplified product(amplicon) remains double stranded, thereby involving little, if any, orno detectable change of signals. While in other embodiment, themeasuring melting profile may be performed when some of the amplifiedproduct is in single-stranded form.

The skilled person will be able to determine, merely from the meltingprofile after amplification (and without comparing the melting profilewith any other melting profiles) whether or not at least one targetnucleic acid has been amplified. For example, FIGS. 1 and 2 illustratethe “flatter” curves obtained after amplification, as compared to themore “S” shaped curves which are characteristic of unconsumed probes.

The method of the invention may further comprise a step (d)

(i) comparing at least two melting profiles obtained in (c) and/or(ii) comparing a melting profile obtained in step (c)

-   -   with a previously-obtained melting profile of the same probes or    -   with a melting profile of the same probes obtained in parallel        at the same time in control reactions, or    -   with a theoretical melting profile of the same probes,        wherein a change in the melting profile provides an indication        of whether or not at least one target nucleic acid has been        amplified in said sample/amplification reaction mixture.

The melting profile may be measured before amplification takes place(pre-amplification melting profile), and/or is measured after completionof amplification (post-amplification melting profile), and/or ismeasured during amplification at each cycle or selected cycles(mid-amplification melting profile),

A pre-amplification melting profile of the probes may be measured in thesame reaction vessel before the start of amplification, or may bemeasured in a separate reaction vessel where no amplification takesplace due to that the reaction mixture lacking one or more ingredientsnecessary for the amplification. Examples of such ingredients include adNTP, a polymerase or a target nucleic acid.

It should be noted that the method of the invention does not necessarilyrequire the pre-amplification melting profile to be measured as part ofthe method of the invention. This profile might be one which ischaracteristic of the probe or combination of probes in question andwhich might have been measured previously and/or stored in a retrievablemanner, for example in a computer-readable format.

Each probe in the reaction mixture will be specific for a particulartarget nucleic acid. Different probes are used which are labelled withthe same label or labels having undistinguishable emission spectra, andthe probes are selected so as to have different melting characteristics,for example different melting profiles or different melting temperatures(T_(m)s).

The melting profile which is obtained in step (c) will be anamalgamation of the melting profiles of the probes which are present inthe amplification reaction mixture. Due to the fact that differentprobes are selected so as to have different melting profiles, theindividual contributions made by each probe to the combined meltingprofile will be separable by those skilled in the art, either manuallyor preferably by computer-implemented means. In this way, the presenceor absence of particular probes, and hence the presence or absence ofparticular target nucleic acids in the sample, can be distinguished fromone another.

Comparison of the pre- and post-amplification melting profiles of eachprobe may identify which feature of the curve that is a signature for aparticular probe has disappeared or has been reduced, thereforeindicating that that particular probe is consumed during amplificationand further indicating that the corresponding target is present in thesample.

It is preferred that the comparison of the melting profiles of probesbefore amplification taking place and after completion of amplificationor after some cycles of amplification is done by a computer program. Thecomputer program determines which probe or how much the probe isconsumed, which is indicative of which target or how much of thecorresponding target is present in a sample.

The melting temperatures (T_(m)) of probes with the same label(s) ordifferent label(s) with indistinguishable emission spectra willgenerally be different from the melting temperatures of each othersimilarly-labelled probe. In one embodiment, multiple probes in a setare each labelled with the same label (or different label(s) withindistinguishable emission spectra) and each probe has a distinctmelting temperature range. In a multiplex assay, when a reactiontemperature rises from the hybridising temperature to a denaturingtemperature, the probe duplex with the lowest T_(m) unwinds first, theprobe duplex with the next highest T_(m) separates next, and the probeduplex with the highest T_(m) denatures last. Concurrently, thefluorescent emission of the label attached to the probe changes inproportion to the rising reaction temperature due to the incrementalmelting of the probe duplex, hence allowing each probe to bedistinguished in the combined melting profile. The shape and position ofa melting curve is a function of GC/AT ratio, length, and the sequenceof the double-stranded portion of the probe.

Preferably, the T_(m)s of probes with the same label(s) or differentlabel(s) with indistinguishable emission spectra are at least 2° C.,preferably at least 3° C., 4° C. or 5° C. different from othersimilarly-labelled probes.

In one aspect of the present invention, real time fluorescencemonitoring of a PCR reaction is used to acquire a probe's melting curvesduring the PCR reaction. The temperature cycles of PCR that driveamplification alternately denature the accumulating product and thelabelled probes at a high temperature, and anneal the primers and atleast one strand of the probe to the product at a lower temperature,thereby consuming some probes. Plotting fluorescence as a function oftemperature as the sample is heated through the dissociation temperatureof probe's internal double stranded portion of the remaining(unconsumed) probe gives a probe's melting curve. Thus continuousmonitoring of fluorescence during a PCR reaction provides a system fordetecting changes of probe concentrations by probe melting profiles.Such a system, particularly a HRM-PCR system, can be used todifferentiate the remaining probes separated by less than 2° C. inmelting temperature.

The invention also provides a method for monitoring a PCR amplificationof at least two nucleic acid targets, said method comprising:

(a) contacting a sample comprising one or more target nucleic acids withan amplification reaction mixture comprising:

-   -   (i) one or more pairs of forward/reverse oligonucleotide        primers, wherein the primer pairs are capable of amplifying one        or more target nucleic acids, if present in the sample, and a        nucleic acid polymerase,    -   (ii) a set of two or more probes, wherein at least one probe        comprises a double stranded portion, which can be molecular        beacon probe, or which may comprise two strands:        -   a first oligonucleotide which comprises a first region which            is substantially complementary to part of one target nucleic            acid and a second region, and        -   at least one second oligonucleotide which comprises a region            which is substantially complementary to a second region of            the first oligonucleotide, such that the first and second            oligonucleotides are capable of forming a double-stranded            portion,    -   wherein each probe comprises a detectable label which is capable        of producing a changeable signal which is characteristic of the        presence or absence of a double-stranded portion between the        first and second oligonucleotides of that probe, and    -   wherein the at least two of the probes comprise the same        detectable label or different detectable labels with        undistinguishable emissions spectra    -   wherein the melting characteristics of the double-stranded        portions of each of such probes are different;        (b) performing an amplification reaction on the        sample/amplification reaction mixture wherein, when a target        nucleic acid is present, the corresponding probes which are        substantially complementary to part of that target nucleic acid        are consumed during the amplification reaction; and

wherein the step (b) further comprises the step (b1) obtaining cycle bycycle fluorescence emissions (FE) at various measuring temperatures(MT), wherein said fluorescence emissions (FE) are baseline correctedfluorescence (dR),

wherein when said amplification reaction mixture comprises “n” probesfor multiplex detection of “n” nucleic acid targets, wherein first probehas a melting temperature of T_(m)1, second probe has a meltingtemperature of T_(m)2, third probe has a melting temperature of T_(m)3,n-th probe has a melting temperature of T_(m)n, whereinT_(m)1>T_(m)2>T_(m)3 . . . >T_(m)n, wherein the percentages of thedouble-stranded form of each probe at a particular temperature ordifferent temperatures are determined experimentally or are calculatedin theory by a computer program, wherein a first fluorescence emissionFEa is obtained at a measuring temperature MTa, at which more than 50%of first probe is in duplex form, second fluorescence emission FEb isobtained at a measuring temperature MTb, at which more than 50% ofsecond probe is in duplex form, n−1 fluorescence emission FE(n−1) isobtained at a measuring temperature MT(n−1), at which more than 50% of(n−1)th probe is in duplex form, n-th fluorescence emission FEn isobtained at a measuring temperature MTn, at which more than 80% of n-thprobe is in duplex form, and optionally a fluorescence emission FE0 isobtained at a measuring temperature MT0, at which no more than 10% offirst probe is in duplex form, wherein n is a positive integer and n 2,

wherein the step (b) may further comprise the step (b2) determiningcycle by cycle Actual Consumed Amount of fluorescence emission fromconsumed probe for each probe, wherein the Actual Consumed Amount offluorescence emission t of k-th probe is depicted as ACA_(k). At aparticular measuring temperature (MTa), wherein a probe has certainpercentage (dska) % in ds (double-strand) form, the fluorescenceemission FE at this measuring temperature MT contributed by first probewill be (ds1a) %*(ACA_(k)), contributed by the second probe will be(ds2a) %*(ACA₂), contributed by the k-th probe will be (dska)%*(ACA_(k)). For example, at 60° C. 70% of probe 1 is in ds form; at 50°C. 80% in ds form. At 60° C. the FE contributed by the probe 1 will be70%*(ACA₁); at 50° C. FE contributed by the probe 1 will be 80%*(ACA₁).If multiple probes are present, the FE will be the total amountcontributed by consumed probes of all probes. The calculation of ActualConsumed Amount (ACA) can use the following formula:

At temperature “a”, the total fluorescence emission will be

FEa=(ACA1)*(ds1a)%+(ACA2)*(ds2a)%+(ACA3)*(ds3a)% . . . +(ACAn)*(dsna)%

At temperature “b”, the total fluorescence emission will be

FEb=(ACA1)*(ds1b)%+(ACA2)*(ds2b)%+(ACA3)*(ds3b)% . . . +(ACAn)*(dsna)%

At temperature “c”, the total fluorescence emission will be

FEc=(ACA1)*(ds1c)%+(ACA2)*(ds2c)%+(ACA3)*(ds3c)% . . . +(ACAn)*(dsna)%

And so on. The individual ACA can be calculated from the above formulas.

wherein the emission amount of each probe is obtained though a computerprogram or is done manually.

The invention also provides a computer software product for use with themethod of the invention adapted, when run on suitable data processingmeans, for comparing melting profiles of probes and/or quantifying areal time PCR amplification of multiplex targets which performs thecalculation of the florescence emission and Actual Consumed Amount(ACA).

Generally, ACA can be calculated manually once the emission values areacquired through a PCR instrument and the percentages of the doublestranded form of each probe at each temperature are known. However, itis frequently desirable to automate the calculation through the use of acomputer system.

In a further embodiment, the invention relates to a computer systemcomprising a computer memory having a computer software program storedtherein, wherein the computer software program, when executed by aprocessor or in a computer, performs methods according to the presentinvention. In a preferred embodiment, a computer program productcomprises a computer memory having a computer software program storedtherein, wherein the computer software program performs a methodcomprising the step of calculating the ACA and/or determination offeatures of melting profiles during amplification or at the endamplification.

As will be appreciated by those skilled in the art, a computer programproduct of the present invention, or a computer software program of thepresent invention, may be stored on and/or executed in a PCR instrumentand used to calculate the amount of each probe.

The invention further provides a kit for assaying for one or morenucleic acid targets, which kit comprises a probe comprising:

-   -   a first oligonucleotide of 15-150 nucleotides which comprises a        first region which is substantially complementary to part of one        target nucleic acid and a second region, and    -   at least one second oligonucleotide of 4-150 nucleotides which        comprises a region which is substantially complementary to the        second region of the first oligonucleotide, such that the first        and second oligonucleotides are capable of forming a        double-stranded portion,    -   wherein each probe comprises a detectable label or detectable        combination of labels which is/are capable of producing a        changeable signal which is characteristic of the presence or        absence of a double-stranded portion between the first and        second oligonucleotides of that probe,        and wherein        (a) the first oligonucleotide of the probe does not comprise a        label, the second oligonucleotide comprises a first label and a        second label, wherein the first label is attached at or near one        end of second oligonucleotide and the second label is attached        at or near the other end of the second oligonucleotide, whereby        when the second oligonucleotide is not hybridised with the first        oligonucleotide, the second oligonucleotide is in a        random-coiled or a stem-loop structure which brings the first        label and second label in close proximity and wherein when the        second oligonucleotide is hybridised with the first        oligonucleotide, the two labels are held away from each other;        or        (b) the first oligonucleotide does not comprise a label and the        second oligonucleotide comprises a label, wherein when the        second oligonucleotide hybridises to the first oligonucleotide        to form the double-stranded portion of the probe, the label is        capable of changing its detectable signal emission relative to        the emission of the label when in the single-stranded form of        the second oligonucleotide; or        (c) the first oligonucleotide of the probe does not comprises a        label, the probe comprises two second oligonucleotides which are        capable of hybridising adjacently or substantially adjacently to        different parts of the second region of the first        oligonucleotide, wherein one of the second oligonucleotides is        attached with a first label, and the other second        oligonucleotide is attached with a second label, such that when        the two second oligonucleotides are hybridised to the first        oligonucleotide, the two labels are brought in close proximity        and one label affects the signal from the other.

The invention also provides the use of a probe as defined in (a)-(c)above in a method of the invention.

The invention also provides the use of a probe comprising

-   -   a first oligonucleotide of 15-150 nucleotides which comprises a        first region which is substantially complementary to part of one        target nucleic acid and a second region, and at least one second        oligonucleotide of 4-150 nucleotides which comprises a region        which is substantially complementary to the second region of the        first oligonucleotide,    -   such that the first and second oligonucleotides are capable of        forming a double-stranded portion,    -   wherein each probe comprises a detectable label or detectable        combination of labels which is/are capable of producing a        changeable signal which is characteristic of the presence or        absence of a double-stranded portion between the first and        second oligonucleotides of that probe,        and wherein    -   (a) the first label is attached to the second region of the        first oligonucleotide and the second label is attached to the        region of the second oligonucleotide which is complementary to        the second region of the first oligonucleotide such that the        first and second labels are brought into close proximity upon        formation of the probe's internal duplex, or    -   (b) the first and second oligonucleotides of the probe are        joined by a linker moiety which comprises nucleotides or a        non-nucleotide chemical linker, allowing the first        oligonucleotide and second oligonucleotide to form a stem-loop        structure, wherein the first and second oligonucleotides are        each labelled such that, when the probe forms an internal        stem-loop structure, the labels are brought into close proximity        and one label affects the signal from the other,        in a method as disclosed herein.

Another aspect of the invention is directed to a method for assaying asample for one or more variant nucleotides on the target nucleic acids,said method comprising:

(a) contacting a sample comprising target nucleic acids with anamplification reaction mixture comprising:

-   -   (i) one or more pairs of forward/reverse oligonucleotide        primers, wherein the primer pairs are capable of amplifying one        or more target nucleic acids, if present in the sample,    -   (ii) at least one pair of probes, wherein first probe in the        pair comprises sequence complementary to the wild-type target        nucleic acid sequence (the normal sequence), second probe in the        pair comprises sequence complementary to the target nucleic acid        sequence containing variant nucleotides (for example, SNP,        mutated nucleotides etc), wherein each probe in the pair        comprises        -   a first oligonucleotide which comprises a first region which            is substantially complementary to part of one target nucleic            acid and a second region, and at least one second            oligonucleotide which comprises a region which is            substantially complementary to the second region of the            first oligonucleotide, such that the first and second            oligonucleotides are capable of forming a double-stranded            portion,        -   wherein each probe in the pair comprise the same second            oligonucleotide,    -   wherein each probe comprises a detectable label or detectable        combination of labels which is/are capable of producing a        changeable signal which is characteristic of the presence or        absence of a double-stranded portion between the first and        second oligonucleotides of that probe, and    -   wherein at least two of the probes comprise the same detectable        label or different detectable labels with undistinguishable        emission spectra and wherein the melting characteristics of the        double-stranded portions between the first and second        oligonucleotides of each of such probes are different;        (b) performing an amplification reaction on the        sample/amplification reaction mixture wherein, when a target        nucleic acid is present, the first oligonucleotides of probes        which are substantially complementary to part of that target        nucleic acid are consumed during the amplification reaction; and        (c) measuring, at least once, the melting profile of any        double-stranded portions between the first and second        oligonucleotides of any unconsumed probes by detecting the        signal(s) from the labels in those probes as a function of        temperature,        wherein the melting profile provides an indication of whether or        not at least one target nucleic acid has been amplified in said        sample/amplification reaction mixture.

The same second oligonucleotide in the pair of the probe may compriseuniversal base or Inosine which corresponds to the variant nucleotide inthe target nucleic acid sequence. The universal base may be3-nitropyrrole 2′-deoxynucleoside, 5-nitroindole, pyrimidine analog orpurine analog. Inosine occurs naturally in the wobble position of theanticodon of some transfer RNAs and is known to form base pairs with A,C and U during the translation process (FIG. 14).

For scanning multiple mutations or SNPs in a target sequence, multiplefirst oligonucleotides of different probes hybridising to differentsites of the same amplified product may be included in a reaction. Theprobes may contain a competing pair of probes, the first probe in thepair hybridises to the wild-type (normal nucleotide) sequence; thesecond probe in the pair hybridises to target sequence containing thevariant (mutated) nucleotides. When the wild-type target sequence ispresent, the probe complementary to the wild-type target sequence isconsumed. When the target sequence having the variant nucleotide ispresent, the probe complementary to the variant target sequence isconsumed. The multiple first oligonucleotides may hybridise to the samestrand of target nucleic acid sequence adjacent to each other or withsome overlapping region between adjacent first oligonucleotides (FIG.14).

The invention further provides a method for assaying a sample for one ormore target nucleic acids, said method comprising:

(a) contacting a sample comprising one or more target nucleic acids withan amplification reaction mixture comprising:

-   -   (i) one or more pairs of forward/reverse oligonucleotide        primers, wherein the primer pairs are capable of amplifying one        or more target nucleic acids, if present in the sample,    -   (ii) a set of two or more probes, wherein at least one probe in        the set comprises a double-stranded portion,    -   wherein at least one probe comprises two oligonucleotides: first        oligonucleotides, which is also referred to as a        target-hybridising oligonucleotide (THO) and second        oligonucleotide, which is also referred to as a partially        complementary oligonucleotide (PCO), THO and PCO are capable of        hybridising to each other, forming a partially double-stranded        probe,    -   wherein each probe in the set comprises a detectable label or        detectable combination of labels which is/are capable of        producing a changeable signal which is characteristic for each        probe, and    -   wherein said two or more probes comprise the same detectable        label or different detectable labels with undistinguishable        emission spectra and wherein the melting characteristics of each        of such probes are different, each probe with double-stranded        portion has a signature melting temperature, whereas        single-stranded probes having no double stranded portion do not        have a signature melting temperature;        (b) performing an amplification reaction on the sample/reaction        mixture under amplification conditions, wherein, when a target        nucleic acid is present, THO which are substantially        complementary to part of that target nucleic acid are hybridised        with the target nucleic acid, therefore being consumed, wherein        the consumption of probes causes changes of detectable signal in        the labels, and the consumed probes are no longer able to form a        double stranded portion if the original probe has a        double-stranded portion; and        (c) measuring, at least once, the melting profile of the        unconsumed probes in the reaction mixture by detecting the        signal(s) from the labels in those probes as a function of        temperature, wherein the presence or absence of melting        characteristics of any probes in the melting profile analysis is        an indication of unconsumption or consumption of that probe,        which further provides an indication of whether or not at least        one target nucleic acid is present in said sample.

wherein THO is complementary to a target sequence, and is labeled with afluorophore and a quencher,

wherein PCO, which is partially complementary to THO, contains modifiedthe 3′ end to prevent its extension, e.g. by attaching a label or aphosphate group,

wherein, if the 3′ end of PCO is attached with a label which is notquencher, for example a phosphate group, fluorescence emission isincreased by hybridisation of THO and PCO, this type of probe is termedplus probe (+THO:PCO),

wherein, if the 3′ end of PCO is attached with a quencher, fluorescenceemission is decreased by hybridisation of THO and PCO, this type ofprobe is termed minus probe (−THO:PCO).

The set of two or more probes may comprise plus probes only. Or the setof two or more probes may comprise minus probes only. Or the set of twoor more probes may comprise mixed plus probes and minus primers. Or theset of two or more probes may comprise mixed single-stranded probe andplus probes or minus probes.

The consumption of probes may be achieved through hybridisation of theTHO to the target sequence, which is followed by the incorporation ofthe THO into the amplified product, or wherein when the THO can beincorporated into the amplified product, the THO is an extendable primeror one of the pair of forward/reverse oligonucleotide primers.

The consumption of probes may be achieved through hybridisation of theTHO to the target sequence, which is followed by degradation of the THO,wherein when the THO is degraded during the reaction, the reactionmixture comprises an enzyme with nuclease activity.

The set of two or more probes comprises at least two probes havingdouble-stranded portions,

wherein a first probe has a melting temperature T_(m)1 in terms of itsdouble-stranded portion,

wherein a second probe has a melting temperature T_(m)2 in terms of itsdouble-stranded portion,

wherein T_(m)1>T_(m)2,

wherein the same labels are independently attached to the first andsecond probes,

wherein a reduction of any melting peak at T_(m)1 and/or T_(m)2 providesindication of consumption of the first and/or second probe(s).

In one embodiment, the amplification reaction mixture may comprises tworelated THO (probes) for assaying single nucleotide polymorphism (SNP),one THO is for one allele, second THO is for second allele. The tworelated THOs for assaying single nucleotide polymorphism (SNP) arecapable of hybridising to the same PCO to form partially double strandedprobes. The two related THOs may be attached with the same label(s). Thetwo related THOs may differ by one nucleotide which is corresponding tothe SNP. When one SNP is present as homozygous, its corresponding THO isconsumed; its signature peak in the melting curve profile is disappearedor reduced. When the SNP is present as heterozygous, both THOs areconsumed; the signature peaks for both alleles in the melting curveprofile are disappeared or reduced.

The invention also provide a kit for assaying for one or more nucleicacid targets, which comprises a set of two or more probes according toany one of the preceding claims comprising:

-   -   at least one probe in the set comprises a double-stranded        portion,    -   wherein said at least one probe comprises two oligonucleotides:        a target-hybridising oligonucleotide (THO) and a partially        complementary oligonucleotide (PCO), THO and PCO are capable of        hybridising to each other, forming a partially double-stranded        probe, wherein each probe in the set comprises a detectable        label or detectable combination of labels which is/are capable        of producing a changeable signal which is characteristic for        each probe, and    -   wherein said two or more probes comprise the same detectable        label or different detectable labels with undistinguishable        emission spectra and wherein the melting characteristics of each        of such probes are different, each probe with a double-stranded        portion has a signature melting temperature, whereas        single-stranded probes having no double stranded portion do not        have a signature melting temperature;    -   wherein THO is complementary to a target sequence, and is        labeled with a fluorophore and a quencher,    -   wherein PCO, which is partially complementary to THO, contains        modified the 3′ end to prevent its extension, e.g. by attaching        a label or a phosphate group,    -   wherein, if the 3′ end of PCO is attached with a phosphate        group, fluorescence emission is increased by hybridisation of        THO and PCO, this type of probe is termed plus probe (+THO:PCO),    -   wherein, if the 3′ end of PCO is attached with a quencher,        fluorescence emission is decreased by hybridisation of THO and        PCO, this type of probe is termed minus probe (−THO:PCO),    -   wherein said set of two or more probes comprises plus probes        only,    -   or said set of two or more probes comprises minus probes only,    -   or said set of two or more probes comprises mixed plus probes        and minus primers.    -   or said set of two or more probes comprises mixed        single-stranded probe and plus probes or minus probes.

In the kit, the set of two or more probes may comprise two related THOs(probes) for assaying single nucleotide polymorphism (SNP), one THO isfor one allele, second THO is for second allele.,

wherein said two related THOs are capable of hybridising the same PCO toform partially double stranded probes,

wherein said two related THOs are attached with the same label(s).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 graphically presents the melting profile of a nucleic acid probeof the present invention before amplification and after amplification inwhich the probe is consumed.

FIG. 2 graphically presents a melting profile of a mixture of nucleicacid probes (probe 1 and 2) of the present invention beforeamplification and after amplification in which one or both of the probesare consumed.

FIG. 3 graphically presents a real-time measurement of an ampliconsynthesis at different temperatures.

FIG. 4 illustrates examples of different probes which may be used in thepresent invention.

FIG. 5 illustrates an example of a method of the present invention,where the first oligonucleotide of the probe is hybridised with a targetnucleic acid sequence.

FIG. 6 illustrates an example of a method of the present invention,where the first oligonucleotide of the probe is incorporated into theamplified product.

FIG. 7 illustrates an example of a method of the present invention,where the first oligonucleotide of the probe is extended and degradedduring the amplification.

FIG. 8 illustrates an example of a method of the present invention,where the first oligonucleotide of the probe is degraded during theamplification.

FIG. 9A graphically presents a melting profile of probe 1 and 2 of thepresent invention before amplification. FIG. 9B presents a meltingprofile of a mixture of probes 1 and 2 at different ratios.

FIG. 10 graphically presents a melting profile of mixture of probes 1and 2 in the 4 reactions before amplification.

FIG. 11 graphically presents a real-time measurement of ampliconsynthesis at different temperatures in the Example.

FIG. 12 graphically presents a melting profile of a mixture of probes 1and 2 in the 4 reactions after amplification.

FIG. 13 illustrates the amplification plot where the emission amount ofthe first probe (K10) is drawn as FE1. The calculated FE1×P plot isdrawn as FE1×P. The amplification plot for the emission amount of thesecond probe (SV40) is shown as FE2−(FE1×P).

FIG. 14 A set of probes labeled with Fam contain sequences complementaryto the wild-type sequence, which have a different Tm; another set ofprobes labeled with Hex contains sequences complementary to the variantsequence of the same target nucleic acid sequence.

FIG. 15 illustrates a method using the set probes described in FIG. 14.

FIG. 16 shows results of an example of triplex amplification. Threeprobes with different Tms are labeled with Fam dye. (A) shows a meltingprofile generated in a tube without adding target DNA. The followingfigures show comparison of melting profile generated in a tube withoutadding target DNA and melting profile generated in a tube with addingtarget DNA. (B) melting profiles generated in a tube with target 2present in the sample. (C) melting profile generated in a tube withtarget 3 present in the sample. (D) melting profile generated in a tubewith targets 2 and 3 present in the sample. (E) melting profilegenerated in a tube with target 1 present in the sample. (F) meltingprofile generated in a tube with targets 1 and 2 present in the sample.(G) melting profile generated in a tube with target 1 and 3 present inthe sample. (H) melting profile generated in a tube with targets 1, 2and 3 present in the sample.

FIG. 17 (A) shows the melting temperature and collection setting upprofile. (B) is melting profiles of probe 1, 2 and the mix of probe 1and 2. (C) is the multicomponent view of the dissociation curve showinghow to estimate of the percentage of the double-stranded form of aprobe.

FIG. 18 is the graphic presentations of the amplification plots forActual Consumed amount and standard curves (Example 4).

FIG. 19. Nucleic acid probe design and its melting profile. (A) Plusprobe consists of a target-hybridising oligonucleotide (THO) labeledwith a Fluorophore and a Quencher, and a partially complementaryoligonucleotide (PCO) without a label. The melting curve of THO:PCOplotted by the first negative derivative of the emission reading versustemperature, reveals a positive value (+THO:PCO). (B) Minus probeconsists of a THO labeled with a Fluorophore and a Quencher and a PCOlabeled with a Quencher at the 3′ end. The melting curve plotted by thefirst negative derivative of the emission reading versus temperature,reveals a negative value (−THO:PCO).

FIG. 20. Design of HPV16 and HPV18 probes and the use of the probes foramplification and melting curve analysis. (A) Hybrids of THO:PCO areformed through a combination of THO and PCO at a ratio of 1:2. (B)Melting profile of the mix of HPV16 and HPV18 probes is plotted as thefirst negative derivative of the emission reading versus temperature.(C) is the graphic presentation of the amplification plot of PCR onthree ten-fold serial dilutions of HPV16 templates. (D) is the samereaction as (C) but is the melting curve plotted as the first negativederivative of the emission reading versus temperature. (E) is thegraphic presentation of the amplification plot of PCR on three ten-timesdilutions of HPV18 templates. (F) is the same reaction as (E) but is themelting curve plotted by the first negative derivative of the emissionreading versus temperature.

EXAMPLES

The present invention is further defined in the following Examples, inwhich parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions. Thus, various modifications of theinvention in addition to those shown and described herein will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims.

The disclosure of each reference set forth herein is incorporated hereinby reference in its entirety.

Example 1

All primers used in the subsequent experiments were synthesized byEUROGENTEC. Amplification primers and probe are:

(SEQ ID NO: 1) K10R266Fam GttcaATTGGGTTTCACCGCGCTTAGTTACA;(SEQ ID NO: 2) K10R266Dab GCGCGGTGAAACCCAATTGAAC; (SEQ ID NO: 3)SV40R1F3FAM ATCAGCCATACCACATTTGTAGAGGTTTTAC; (SEQ ID NO: 4) SV40R1F3DabCAAATGTGGTATGGCTGAT; (SEQ ID NO: 5) K10F155 CTCTGCTGACTTCAAAACGAGAAGAG;(SEQ ID NO: 6) SV40Rea1R CCATTATAAGCTGCAATAAACAAGTTAACAAC

Primer/probe K10R266Fam (the first oligonucleotide) is labelled at the5′ end with FAM. K10R266Dab (the second oligonucleotide) contains DABCYLat the 3′ end. K10R266Fam and K10R266Dab can form double-strandedportion as:

(SEQ ID NO: 1) Fam- 5′GTTCAATTGGGTTTCACCGCGCTTAGTTACA3′ (SEQ ID NO: 2)DABCYL- 3′CAAGTTAACCCAAAGTGGCGCG5′

The above hybrid is referred to as probe K10R266. Primer/probeSV40R1F3FAM (the first oligonucleotide) is labelled at the 5′ end withFAM. SV40R1F3Dab (the second oligonucleotide) contains DABCYL at the 3′end. SV40R1F3FAM and SV40R1F3Dab can form double-stranded portion as:

(SEQ ID NO: 3) Fam- 5′ATCAGCCATACCACATTTGTAGAGGTTTTAC3′ (SEQ ID NO: 4)DABCYL- 3′TAGTCGGTATGGTGTAAAC5′This hybrid is referred to as probe SV40R1F3.

The first oligonucleotide and the second oligonucleotide were combinedat various ratios, typically 1:3 to form a partially double-strandedlinear DNA probe. In the absence of target, the formation of the firstand the second oligonucleotide hybrid brings the quencher and thefluorophore into close proximity, efficiently quenching the fluorescentsignal. In the presence of the target, the first oligonucleotidepreferentially hybridises to the target sequence and incorporates intothe amplicon. As a result, the quencher is separated from thefluorophore resulting in an increase in fluorescence emission.

Primer pair K10R266Fam and K10F155 amplifies a 110 bp product in thepresence of a K10 target sequence. SV40R1F3FAM and SV40RealR amplifies a125 bp product in the presence of an SV40 target sequence.

Example 2

Melting profile experiments were performed as follows: The thermalstability of the probes was characterized in melting profile experimentswhere fluorescence emission was measured at temperatures ranging from 40to 90° C. Melting temperature T_(m) is defined as the characteristictemperature where the first and the second oligonucleotide duplexdissociate.

Each melting profile was measured in a tube in a 20 μl reactioncontaining PCR buffer (1× ThermoPol reaction buffer, New EnglandBioLabs). Thermal cycling was performed in an Mx3005p quantitative PCRsystem (Stratagene) with the following cycling conditions: 1 cycle ofdenaturation at 90° C. for 3 min; 50 cycles of 30 second holding at arange of temperatures from 40 to 90° C. with a 1° C. increment percycle. Fluorescence measurements were recorded during each 30 secondhold of the 50 cycles.

Melting profiles were determined for probe K10R266 (probe 1) and probeSV40R1F3 (probe 2) by monitoring fluorescence at temperatures rangingfrom 90 to 40° C. K10R266 has a T_(m) of 72° C.; SV40R1F3 has a T_(m) of62° C. (FIG. 9A).

Melting profiles were determined for a series of mixtures of probeK10R266 and probe SV40R1F3:

Sample 1 contains 0.5 μM of K10R266 and 0.5 μM of SV40R1F3.

Sample 2 contains 0.5 μM of K10R266 and 0.25 μM of SV40R1F3.

Sample 3 contains 0.5 μM of K10R266 and 0.125 μM of SV40R1F3.

Sample 4 contains 0.5 μM of K10R266 and 0.0625 μM of SV40R1F3.

Sample 5 contains 0.5 μM of K10R266 and 0.003125 μM of SV40R1F3. Theprofiles are shown in FIG. 9B.

A combination of probes K10R266 and SV40R1F3 were tested in real-timePCR assays using plasmids DNA containing K10 and SV40 sequence astemplates.

A master reaction mixture was made containing 0.5 μM of K10R266 and 0.5μM of SV40R1F3 (combination of first oligonucleotide and secondnucleotide in 1:3 ratio mix) and standard PCR ingredients (NEB).Amplification reactions were performed in Stratagene Mx3005 real-timePCR system with the following cycling conditions:

-   -   1. Before amplification, melting profile: 50 cycles of 30 second        holding at a range of temperatures from 90 to 40° C. with a        1° C. decrement per cycle, fluorescence measurements were        recorded during each 30 second hold of the 50 cycles.    -   2. Amplification: 30 cycles of 94° C. 20 s; 63° C. 30 s; 51° C.        30 s; 72° C. 30 s; fluorescence measurements were recoded during        the read steps 63° C., 51° C. and 72° C.    -   3. Post-amplification melting profile: 50 cycles of 30 second        holding at a range of temperatures from 40 to 90° C. with a        1° C. increment per cycle, fluorescence measurements were        recorded during each 30 second hold of the 50 cycles.

Four reactions were set up: reaction 1 contains K10 template; reaction 2contains SV40 template; reaction 3 contains both K10 and SV40 templates;reaction 4 contains no template.

The pre-amplification melting profiles for four reactions are shown inFIG. 10. Fluorescence emission was collected at 63° C. duringamplification is shown in FIG. 11A. Fluorescence emission was collectedat 51° C. during amplification is shown in FIG. 11B. Fluorescenceemission was collected at 72° C. during amplification is shown in FIG.11C. The post-amplification melting profiles for four reactions areshown in FIG. 12.

Comparison of the pre-amplification and post-amplification meltingprofiles indicated the following:

-   -   In reaction 1 the K10R266 probe is consumed and the profile is        the signature of SV40R1F3 probe.    -   In reaction 2 the SV40R1F3 probe is consumed.    -   In reaction 3, both K10R266 and SV40R1F3 probes are consumed.    -   In reaction 4, no probe is consumed and the pre- and        post-amplification profiles are similar.

Cycle by cycle fluorescence emissions FE were obtained at threemeasuring temperatures: MT 72° C., 63° C. and 51° C.

A first fluorescence emission FE1 is obtained at a measuring temperature63° C., at which no more than 10% of second probe (SV40R1F3) is induplex form (the internal double-stranded form of the probe); secondfluorescence emission FE2 is obtained at a measuring temperature 51° C.,at which more than 95% of two probes are in duplex form, and optionallya fluorescence emission FE0 is obtained at a measuring temperature 72°C., at which no more than 10% of first probe (K10R266) is in duplexform.

In the amplification reactions there are two probes for two targetsequences K10 and SV40. At temperature 72° C., 10% of the K10R266 probeis in duplex form, 0% of the SV40R1F3 probe is in duplex form. At 63°C., 90% of the K10R266 probe is in duplex form, 5% of SV40R1F3 probe isin duplex form. At 51° C., more than 98% of all probes are in duplexform.

The first fluorescence emission is collected at 63° C., which is FE1;the second fluorescence emission is collected at 51° C., which is FE2.

Probe 1 (K10) Probe 2 (SV40) fluorescence ds % ACA1 ds % ACA2 emission72° C. 10 10% ACA1 0  0% ACA2 63° C. 90 90% ACA1 5  5% ACA2 FE1 51° C.98 98% ACA1 98 98% ACA2 FE2 FE1 = 90% * ACA1 + 5% * ACA2 FE2 = 98% *ACA1 + 98% * ACA2

Assuming 5%*ACA2 is negletable, ACA1=FE1/90%; ACA2=FE2/98%-FE1/90%. ACA1is the actual consumed amount of probe 1; ACA2 is the actual consumedamount of probe 2.

The amplification plot for the actual consumed amount of the first probe(K10) is drawn as ACA1. The amplification plot for the actual consumedamount of the second probe (SV40R1F3) is shown as ACA2 (FIG. 13).

The calculation of the actual consumed amount can be performed manually.The calculation can also be performed through a computer program orsoftware. To expedite the quantification, software was designed tomanage emission data from the multiplex real-time PCR and performappropriate calculations. The software had other functions, such asmanual selection of the Ct and subtraction of blanks.

The software was implemented in Visual Basic for applications (VBA) asan Addin for Microsoft Excel. The source code was organized in two mainmodules. One module contained all the “utility” functions such asmathematical functions, functions to generate arrays from emission datapresent in the Excel sheets, functions to print result data and labels,functions to handle errors or template and functions to generate chartsof a certain types. The second module contained the functions to controlthe flow of the program. This module contained all the functions makingpossible the interaction with the user, such as menu selections, barslicing, inclusion/exclusion of data in the standard curve.

Example 3 Amplification Primers and Probe are

For target 1 (K10) (SEQ ID NO: 1) K10R266FamGttcaATTGGGTTTCACCGCGCTTAGTTACA; (SEQ ID NO: 2) K10R266DabGCGCGGTGAAACCCAATTGAAC; (SEQ ID NO: 5) K10F155CTCTGCTGACTTCAAAACGAGAAGAG; For target 2 (SV40) (SEQ ID NO: 3)SV40R1F3FAM ATCAGCCATACCACATTTGTAGAGGTTTTAC; (SEQ ID NO: 4) SV40R1F3DabCAAATGTGGTATGGCTGAT; (SEQ ID NO: 6) SV40Rea1RCCATTATAAGCTGCAATAAACAAGTTAACAAC; For target 3 (Jak2) (SEQ ID NO: 11)JKR3Fam AACAGATGCTCTGAGAAAGGCATTAGA; (SEQ ID NO: 12) JKR3FDabFCTCAGAGCATCTGTT; (SEQ ID NO: 13) JKF2 GCATCTTTATTATGGCAGAGAGAA.

The oligonucleotide K10R266Fam is an amplification primer, in the sametime the oligonucleotide K10R266Fam is the first oligonucleotide of theprobe 1. K10R266Fam (the first oligonucleotide) is labelled at the 5′end with FAM. K10R266Dab (the second oligonucleotide) contains DABCYL atthe 3′ end. K10R266Fam and K10R266Dab can form double-stranded portion.The hybrid of K10R266Fam and K10R266Dab is referred to as probe 1.

The oligonucleotide SV40R1F3FAM is an amplification primer, in the sametime the oligonucleotide SV40R1F3FAM is the first oligonucleotide of theprobe 2. SV40R1F3FAM (the first oligonucleotide) is labelled at the 5′end with FAM. SV40R1F3Dab (the second oligonucleotide) contains DABCYLat the 3′ end. SV40R1F3FAM and SV40R1F3Dab can form double-strandedportion. The hybrid of SV40R1F3FAM and SV40R1F3Dab is referred to asprobe 2.

The oligonucleotide JKR3Fam is an amplification primer, in the same timethe oligonucleotide JKR3Fam is the first oligonucleotide of the probe 3.JKR3Fam (the first oligonucleotide) is labelled at the 5′ end with FAM.JKR3FDabF (the second oligonucleotide) contains DABCYL at the 3′ end.JKR3Fam and JKR3FDabF can form double-stranded portion. The hybrid ofJKR3Fam and JKR3FDabF is referred to as probe 3.

The first oligonucleotide and the second oligonucleotide were combinedat various ratios, typically 1:2-1:4 to form a partially double-strandedlinear DNA probe. In the absence of target, the formation of the firstand the second oligonucleotide hybrid brings the quencher and thefluorophore into close proximity, efficiently quenching the fluorescentsignal. In the presence of the target, the first oligonucleotidepreferentially hybridises to the target sequence and incorporates intothe amplicon. As a result, the quencher is separated from thefluorophore resulting in an increase in fluorescence emission.

Primer pair K10R266Fam and K10F155 amplifies a 110 bp product in thepresence of a K10 target sequence. SV40R1F3FAM and SV40RealR amplifies a125 bp product in the presence of an SV40 target sequence. PrimersJKR3Fam and JKF2 amplifies a 222 bp product in the presence of Jak2target sequence.

Melting profile analysis of probe 1, probe 2, probe 3 and mixed probe 1,2 and 3 were performed using the Stratagene Mx3005 real-time PCR machine(FIG. 16). The thermal profile was based on the dissociation curveanalysis software parameters: heat at 70° C. for 30 sec, cool to 40° C.hold for 30 second, and then slowly increase the temperature to 94° C.,the fluorescence emission data is continually collected during therising temperatures. The first negative derivative of the emissionreading with respect to temperature is plotted against temperature toform curves, and each peak of the curve corresponds to the actual T_(m)of the probe.

Singleplex, doubleplex and triplex amplifications were performed usingthe same reaction master mix, but in the presence of one target, or twotargets or all three targets.

A master reaction mixture was made containing 0.1 μM of firstoligonucleotides of each of the three probes, 0.4 μM of secondoligonucleotides of each of the three probes, 0.2 μM of primers (not thefirst oligonucleotide of probe) of each target, and standard PCRingredients (NEB). Amplification reactions were performed in StratageneMx3005 real-time PCR system with the following cycling conditions:

-   -   1. Amplification: 40 cycles of 94° C. 15 s; 63° C. 20 s; 50° C.        20 s; 55° C. 20 s 63° C. 20 s; 68° C. 20 s; 72° C. 20 s;        fluorescence measurements were recorded during the read steps        50° C., 55° C., 63° C., 68° C., and 72° C.    -   3. Post-amplification melting profile: after last cycle at        72° C. for 20 sec, cool to 40° C. hold for 30 second, and then        slowly increase the temperature to 78° C., the fluorescence        emission data is continually collected during the rising        temperatures.

Eight reactions were set up: reaction (A) contains no template; reaction(B) contains target 2 template; reaction (C) contains target 3 template;reaction (D) contains target 2 and 3; reaction (E) contains target 1;reaction (F) contains targets 1 and 2; reaction (G) contains targets 1and 3; reaction (H) contains targets 1, 2 and 3. The post-amplificationmelting profiles for eight reactions are shown in FIG. 16.

Comparison of the post-amplification melting profiles in the presence oftargets and without target indicated the following (FIG. 16):

In reaction A the no probe is consumed and the melting profile is thesignature of the mixture of all three probes.

In reaction B the probe 2 is consumed.

In reaction C, probe 3 is consumed.

In reaction D, probes 2 and 3 are consumed.

In reaction E, probe 1 consumed.

In reaction F, probes 1 and 2 are consumed.

In reaction G, probes 1 and 3 are consumed.

In reaction H, probes 1, 2 and 3 are consumed.

Cycle by cycle fluorescence emissions FE were obtained at five measuringtemperatures: MT 50° C., 55° C., 63° C., 68° C., and 72° C. The ActualConsumed Amount for each probe may be calculated by the formulaFEa=(ACA1)*(ds1a) %+(ACA2)*(ds2a) %+(ACA3)*(ds3a) % . . . +(ACAn)*(dsna)%.

Example 4 Amplification Primers for Target 1 (K10)

(SEQ ID NO: 5) K10F155 CTCTGCTGACTTCAAAACGAGAAGAG; (SEQ ID NO: 21)K10R14 CCTGAGGGTTAAATCTTCCCCATTGA

Probe for target 1 (referred to as probel) includes: firstoligonucleotide K10R266Famph GTTCAATTGGGTTTCACCGCGCTTAGTTACA (SEQ ID NO:7), which 5′ end is attached with Fam; 3′ end is attached with aphosphate group instead of 3′-OH; and second oligonucleotide K10R266DabGCGCGGTGAAACCCAATTGAAC (SEQ ID NO: 2), which 3′ end is attached withDABCYL.

Amplification Primers for Target 2 (SV40)

(SEQ ID NO: 8) dsredendF2 GTAAGATCCACCGGATCTAGATAAC; (SEQ ID NO: 9)sv40testR GGGAGGTGTGGGAGGTTTTTTAAAG.

Probe for target 2 (referred to as probe 2) includes: firstoligonucleotide SV40R1F3FAPh ATCAGCCATACCACATTTGTAGAGGTTTTAC (SEQ ID NO:10), which 5′ end is attached with Fam; 3′ end is attached with aphosphate group instead of 3′-OH; and second oligonucleotide SV40R1F3DabCAAATGTGGTATGGCTGAT (SEQ ID NO: 4), which 3′ end is attached withDABCYL.

The first oligonucleotide and the second oligonucleotide were combinedat various ratios, typically 1:2-1:4 to form a partially double-strandedlinear DNA probe. In the absence of target, the formation of the firstand the second oligonucleotide hybrid brings the quencher and thefluorophore into close proximity, efficiently quenching the fluorescentsignal. In the presence of the target, the first oligonucleotidepreferentially hybridises to the target sequence. As a result, thequencher is separated from the fluorophore resulting in an increase influorescence emission.

The first oligonucleotides in probe 1 and 2 are modified to containblocked 3′ end, so that it cannot be extended. However, when the firstoligonucleotide binds to the target sequence, it can be degraded by the5′ nuclease activity of a polymerase. The degradation of the firstoligonucleotide of the probe (the consumed probes) results in decreaseof the number of first oligonucleotide available to bind with the secondoligonucleotides of probe, thereby increasing the fluorescence signalwhen measured at appropriate temperature.

Melting profile analysis of probe 1, probe 2, and mixed probe 1 and 2was performed using the Stratagene Mx3005 real-time PCR machine (FIG.17). The thermal profile was based on the dissociation curve analysissoftware parameters (FIG. 17A): heat at 72° C. for 30 sec, cool to 40°C. hold for 30 second, and then slowly increase the temperature to 94°C., the fluorescence emission data is continually collected during therising temperatures. The first negative derivative of the emissionreading with respect to temperature is plotted against temperature toform curves, and each peak of the curve corresponds to the actual T_(m)of the probe. The probe 1 has a peak at 67° C. as Tm; the probe 2 has apeak at 59° C. as Tm (FIG. 17B).

The percentages of double stranded form and single stranded form of eachprobe were estimated in the following table. The actual calculation canalso be done by computer software.

probe 1 probe 2 ds % ss % ds % ss % 71° C. 30 70  0 100  69° C. 40 60  0100  67° C. 50 50  1 99 65° C. 65 35  3 97 62° C. 75 25  5 95 61° C. 8515 25 75 59° C. 95  5 50 50 57° C. 97  3 65 35 55° C. 98  2 75 25 54° C.100   0 85 15 52° C. 100   0 100   0

The estimation (calculation) can be done based on the multi-componentview of the dissociation curve (the Fluorescence R versus temperature,FIG. 17C). The base line was assumed as 100% double-stranded, which hasa R as 9000. The R at a temperature (100% single-stranded) is 24500. Attemperature 62° C., the R is 13000. The difference of the fluorescencevalues between 62° C. and base line dR=13000−9000=4000. The percentageof double-stranded probe at 62° C. is estimated as 4000/(24500−9000)which is 25.8%.

Multiplex Real-Time PCR and Standard Curve Analysis

Primer-probe master mix was set up as follows: the primers and probeswere mixed to a final concentration 0.4 μM of probes and 0.6 μM ofprimers, which creates a 2× primer-probe master mix.

The reaction mix was created by combining equal amount of 2×primer-probe master mix and 2× TaqMan® Gene Expression Master Mix(Applied Biosystem, cat. No 4369514).

Template DNAs containing target 1, target 2, and mixed target 1 and 2were serially diluted as follows: 1, 0.1, 0.01, 0.001, 0.0001, 0.00001,0.000001.

The singleplex PCRs were performed using DNA sample containing target 1(k10) or target 2 (SV40). Doubleplex PCR was performed using DNA samplecontaining mixture of target 1 (k10) and target 2 (SV40).

The thermal profile was: 95° C. for 8 min 30 sec; 40 cycles of 94° C. 10s; 66° C. 20 s; 63° C. 20 sec; 54° C. 30 s; 52° C. 20 s; 61° C. 20 s;62° C. 20 s; 68° C. 20 s; fluorescence measurements were recoded duringthe read steps 66° C., 63° C., 54° C.; 52° C.; 61° C.; 62° C.; 68° C.

Fluorescence emission (dR) at 62° C. was chosen as FE1; Fluorescenceemission (dR) at 52° C. was chosen as FE2. Based on the table above andthe formula for calculation of Actual Consumed Amount (ACA) in thedescription and claims, we had followings:

At 62° C., FE1=0.75*(ACA1)+0.05*ACA2  (1)

At 52° C., FE2=100%*ACA1+100%*ACA2  (2)

According to (1) and (2),

ACA1=(FE1−0.05*FE2)/0.7

ACA2=(0.75*FE2−FE1)/0.7

For the doubleplex reaction where both targets are present, The FE1 andFE2 were obtained. The calculated ACAs for the target 1 were obtainedusing ACA1=(FE1−0.05*FE2)/0.7

The graphic presentation of the amplification plots for ACA1 is shown inFIG. 18A. The standard curve is shown in FIG. 18B.

The calculated ACAs for the target 2 were obtained usingACA2=(0.75*FE2−FE1)/0.7 The graphic presentation of the amplificationplots for ACA2 is shown in FIG. 18C. The standard curve is shown in FIG.18D.

If only target 1 is present in the reaction, the graphic presentation ofthe amplification plots for ACA1 is shown in FIG. 18E, the amplificationplots for ACA2 is shown in FIG. 18G. The results show that because onlytarget 1 is present in the reaction, the ACA1 reveal the normalamplification curve and normal standard curve (FIG. 18 F), whereas theACA2 reveal the background curve which demonstrates there is no signalfor target 2.

If only target 2 is present in the reaction, the graphic presentation ofthe amplification plots for ACA1 is shown in FIG. 18H, the amplificationplots for ACA2 is shown in FIG. 18I. The results show that because onlytarget 2 is present in the reaction, the ACA2 reveal the normalamplification curve and normal standard curve (FIG. 18J), whereas theACA1 reveal the background curve which demonstrates there is no signalfor target 1.

Example 5 Amplification Primers for Target 1 (K10)

(SEQ ID NO: 5) K10F155 CTCTGCTGACTTCAAAACGAGAAGAG; (SEQ ID NO: 21)K10R14 CCTGAGGGTTAAATCTTCCCCATTGA

Probe for target 1 (referred to as probel) includes: firstoligonucleotide K10R266Famph GTTCAATTGGGTTTCACCGCGCTTAGTTACA (SEQ ID NO:7), which 5′ end is attached with Fam; 3′ end is attached with aphosphate group instead of 3′-OH; and second oligonucleotide K10R266DabGCGCGGTGAAACCCAATTGAAC (SEQ ID NO: 2), which 3′ end is attached withDABCYL.

Amplification Primers for Target 2 (SV40)

(SEQ ID NO: 8) dsredendF2 GTAAGATCCACCGGATCTAGATAAC; (SEQ ID NO: 9)sv40testR GGGAGGTGTGGGAGGTTTTTTAAAG.

Probe for target 2 (referred to as probe 2) includes: firstoligonucleotide SV40R1F3FAPh ATCAGCCATACCACATTTGTAGAGGTTTTAC (SEQ ID NO:10), which 5′ end is attached with Fam; 3′ end is attached with aphosphate group instead of 3′-OH; and second oligonucleotide SV40R1F3DabCAAATGTGGTATGGCTGAT (SEQ ID NO: 4), which 3′ end is attached withDABCYL.

Amplification Primers for Target 3 (Jak2)

(SEQ ID NO: 14) JknewF8 GTGGAGACGAGAGTAAGTAAAACTACA; (SEQ ID NO: 15)JKnewR8 CTCCTGTTAAATTATAGTTTACACTGACA;

Probe for target 3 (referred to as probe 3) includes: firstoligonucleotide JKR3FamPh AACAGATGCTCTGAGAAAGGCATTAGA (SEQ ID NO: 16),which 5′ end is attached with Fam; 3′end is attached with a phosphategroup instead of 3′-OH; and second oligonucleotide JKR3FDabFCTCAGAGCATCTGTT (SEQ ID NO: 12), which 3′ end is attached with DABCYL.

Amplification primers for target 4 (Kras):

(SEQ ID NO: 17) KR12GVF1B GTCACATTTTCATTATTTTTATTATAAGGCCTGC;(SEQ ID NO: 18) KR12GVR12As GATCATATTCGTCCACAAAATGATTC.

Probe for target 4 (referred to as probe 4) includes: firstoligonucleotide KR12GVFamPh GAATATAAACTTGTGGTAGTTGGAGCTGT (SEQ ID NO:19), which 5′ end is attached with Fam; 3′ end is attached with aphosphate group instead of 3′-OH; and second oligonucleotideKR12GVFamDab CACAAGTTTATATTC (SEQ ID NO: 20), which 3′ end is attachedwith DABCYL.

The first oligonucleotide and the second oligonucleotide were combinedat various ratios, typically 1:2-1:4 to form a partially double-strandedlinear DNA probe. In the absence of target, the formation of the firstand the second oligonucleotide hybrid brings the quencher and thefluorophore into close proximity, efficiently quenching the fluorescentsignal. In the presence of the target, the first oligonucleotidepreferentially hybridises to the target sequence. As a result, thequencher is separated from the fluorophore resulting in an increase influorescence emission.

The first oligonucleotides in all probes are modified to contain blocked3′ end, so that it cannot be extended. However, when the firstoligonucleotide binds to the target sequence, it can be degraded by the5′ nuclease activity of a polymerase. The degradation of the firstoligonucleotide of the probe (the consumed probes) results in decreaseof the number of first oligonucleotide available to bind with the secondoligonucleotides of probe, thereby increasing the fluorescence signalwhen measured at appropriate temperature.

Multiplex Real-Time PCR and Standard Curve Analysis

Primer-probe master mix was set up as follows: the primers and probeswere mixed to a final concentration 0.4 μM of probes and 0.6 μM ofprimers, which creates a 2× primer-probe master mix.

The reaction mix was created by combining equal amount of 2×primer-probe master mix and 2× TaqMan® Gene Expression Master Mix(Applied Biosystem, cat. No 4369514).

The thermal profile was: 95° C. for 8 min 30 sec; 40 cycles of 94° C. 10s; 66° C. 20 s; 63° C. 20 sec; 54° C. 30 s; 49° C. 20 s; 55° C. 20 s;61° C. 20 s; 68° C. 20 s; fluorescence measurements were recoded duringthe read steps 66° C., 63° C., 54° C.; 49° C.; 55° C.; 61° C.; 68° C.Various combinations of targets present in the reaction were used.

Example 6

The principles of this novel method are as follows: Firstly, for eachtarget we design a probe consisting of two oligonucleotides: atarget-hybridising oligonucleotide (THO) and a partially complementaryoligonucleotide (PCO). THO and PCO are capable of hybridising to eachother, forming a partially double-stranded probe (FIG. 19). Due to thedouble-stranded regions, each probe has unique melting properties, whichare mainly characterised by its melting temperature T_(m). THO, which iscomplementary to a target sequence, is labeled with a fluorophore (forexample FAM) at the 5′ end and a quencher (for example BHQ1) at the 3′end. Extension from PCO, which is undesirable, is blocked by modifyingthe 3′ end, e.g. by attaching a label or a phosphate group. If aphosphate group is used (FIG. 19A and FIG. 20A), fluorescence emissionis increased by hybridisation of THO and PCO. As the derivative meltingplots shows a positive value (FIG. 19A), this type of probe is termedplus probe (+THO:PCO). If PCO is labeled with a quencher at the 3′ end(FIG. 19B), fluorescence emission is decreased by hybridisation of THOand PCO. This is because formation of the THO:PCO hybrid brings thequencher and

the fluorophore into close proximity, efficiently quenching thefluorescent signal. The derivative melting plots shows a negative value(FIG. 19B), thus this type of probe is termed minus probe (−THO:PCO).

In a multiplex PCR reaction using this system, there are at least twoprobes for two different targets, which are labeled with the samefluorophore. To distinguish the different probes with the same labels,each probe is designed to have a unique T_(m) which can be recognised ina melting curve analysis. In a proof of principle experiment, wedesigned two probes: one for HPV16 sequence and one for HPV18 sequence,which are both plus probes, with T_(m)(HPV16)=46° C. andT_(m)(HPV18)=37° C. (FIGS. 20A and 20B). In a multiplex reaction, probescan be mixed plus and minus probes. It is also possible that one of theprobes may be a single-stranded probe without a typical T_(m).

Secondly, the target-hybridising oligonucleotide (THO) capable ofhybridising to the target sequence, can be consumed duringamplification. In the absence of a target, THO is not consumed, thusremains in the same concentration throughout the reaction. The meltingprofile of the THO:PCO hybrid does not change. In the presence of atarget, the THO hybridises to the target sequences and, as a result, isconsumed and therefore the concentration decreases. The melting peak ofthe THO:PCO hybrid is reduced or disappears in the melting curveanalysis.

The key feature of consumption of THO can be achieved by any of severalmethods. For example, THO may be incorporated into a PCR product, whereTHO acts as a primer. In this study, we selected the THO to work as aTaqMan probe, which is degraded during the amplification (Holland et al.1991). The degradation of THO, like TaqMan-based real-time PCR, resultsin the increase of fluorescence signal, which can be monitored duringPCR. In the mean time, during the melting curve analysis, thedegradation of THO results in the decrease or disappearance of itssignature melting peak, indicating that the corresponding probe is beingconsumed, which in turn points out which target is amplified.

To test this system, we designed probes and primers for detecting thetwo most virulent and high risk HPV strains: HPV16 and HPV18 in a singledetection channel in a real-time PCR machine. Two plus probes weredesigned, targeting the E6/E7 region of the HPV genome sequence (FIG.20A). Forward and reverse primers were designed to be upstream anddownstream of the probe-binding region. A THO and a PCO were combined ata ratio of 1:2 to form a partially double-stranded DNA probe. In thepresence of the target, the THO preferentially hybridizes to targetsequences and is cleaved by the 5′ nuclease activity of Taq polymeraseduring amplification, resulting in an increase of fluorescence emission.After amplification, a melting curve analysis is performed, wherebyfluorescence and hence the level of THO:PCO hybrids is measured across arange of temperatures.

Real time PCR was performed to test the design of the HPV16 and HPV18probes/primers. Probe hybrids THO:PCO were pre-tested in a melting curveanalysis to determine the fluorescence levels, so that when they weremixed together, they had similar heights of melting peaks (FIG. 20B).The final concentration of HPV16THO and HPV18THO were approximately 200μM. Five ten-fold serial dilutions of template plasmids containing thetarget HPV sequence, and a no DNA negative control were PCR amplified ina Stratagene MX3005P real-time PCR machine, and fluorescence emissionwas collected in the FAM channel. Typical real-time PCR amplificationplots for each series of template dilutions are shown in FIG. 20 (panelC—HPV16, panel E—HPV18, panel G—HPV16+HPV18). Melting profiles wereobtained after amplification. Compared with the negative control, whenthe target HPV16 is present, the melting peak at 46° C. in the meltingcurve has disappeared (FIG. 20D). When the target HPV18 is present, themelting peak at 37° C. has disappeared (FIG. 20F). When both targetsHPV16 and HPV18 are present, the two melting peaks have both disappeared(FIG. 20H). The results clearly demonstrate that HPV16 or HPV18 or bothHPV16+HPV18 can be detected individually and distinguished, even in thesame detection channel.

We next determined whether more targets could be detected in a singledetection channel. Four minus probes labeled with FAM dye were designedfor HPV16, HPV31, HPV52 and HPV59 sequences. The probes' THOs arecomplementary to the conserved L1 region of the HPV genome sequences.After amplification, melting profiles were obtained. Compared againstthe negative control, individual HPV target sequences can be correctlydistinguished. Similarly, four other minus probes labeled with HEX dyewere designed, targeting the conserved L1 region of the HPV genomesequences, which could detect HPV18, HPV39, HPV58 and HPV68 sequences.Results show that these four HPV sequences can be correctly genotyped.

A combination of plus and minus probes were also tested for detectingHPV sequences. Two minus probes labeled with Texas Red dye, targetingthe L1 region of HPV33 and HPV45 sequences, and one plus probe labeledwith the same Texas Red dye, targeting the L1 region of HPV35 sequencewere included in a PCR reaction. After PCR amplification, a meltingcurve analysis was performed. Compared against the no DNA control, inthe presence of each target, its corresponding melting peak eitherreduced or disappeared. We next tested if a combination of minus probeand single-stranded probe could also be used. Two minus probes labeledwith Cy5 dye, targeting the L1 region of HPV56 and HPV66 sequences, andone single stranded probe labeled with the same Cy5 dye, targeting theL1 region of HPV51 sequence were included in a PCR reaction. After PCRamplification, a melting curve analysis was performed. Compared againstthe no DNA control, in the presence of HPV56 or HPV66, its correspondingmelting peak either reduced or disappeared. However, when the targetHPV51 template was present, the melting peaks for HPV56 and HPV66 didnot change, but the whole fluorescence signal increased. The increase ofthe whole fluorescence signal is reflected by proportionally lifting upits melting curve in comparison with the negative control. Theamplification plots also show a normal amplification curve, which is dueto the presence of the HPV51 sequence. This demonstrated that anindividual HPV sequence can be distinguished by using a combination ofmultiple minus probes and one single-stranded probe.

These 14 probes (targeting the L1 region) were mixed together and usedin a single PCR amplification. Of all the 14 high risk HPV sequences,individual sequences were detected and distinguishable in a one-step,closed tube reaction. Our results demonstrate that it may be possible toovercome the current one channel—one target limitation. In accordancewith this method in a multiplex PCR, either real-time monitoring orend-point detection, a greater number of target sequences can beanalysed in a single closed tube by designing sets of probes thathybridise to different target sequences and have different meltingtemperatures. If a target sequence is present, its corresponding probeis consumed. The target can then be determined based on the comparisonof the melting profiles of the probes before and after the reactions.Advantageously, the different probes in a set can be attached with thesame label, allowing for monitoring at a single emission wavelength. Ourmethod provides a significant improvement over the current closed-tubemultiplex PCR technology, allowing for a 2-4 fold increase in thecapacity of targets being analysed in the current instruments.

In the present study, the method is useful for detecting the presence orabsence of a target or multiple targets. It can be performed in anordinary PCR machine, then genotyped for the targets by melting curveanalysis. This is the closed-tube end-point detection approach. Usingthis method eliminates the need for an expensive real-time PCR machine,which in turn is cost saving as there is no need to pay high royalty fora real-time PCR license. It also avoids the need to open the tube forgel analysis. If quantitative data is required, the method can beperformed in a real-time PCR machine by monitoring fluorescence signalin real-time. In a single detection channel, where multiple targets aredetected, the quantity of one of these targets that is present can beprecisely determined. However, if more than one of these targets arepresent, the quantitation data is obtained collectively for thecombination of the multiple targets from one detection channel, orindividual quantitative data can be obtained by the method described inthis invention.

Methods

The target-hybridising oligonucleotides (THOS) were synthesized with afluorescein (FAM) label at the 5′ end and a BHQ1 at the 3′ end. Thenucleotide sequence for HPV16 THO is: 5′ TTCAGGACCCACAGGAGCGACCC 3′. Thenucleotide sequence for HPV18 THO is: 5′ AGCCCCAAAATGAAATTCCGGTTGACC 3′.Partially complementary oligonucleotides (PCOs) were synthesized withthe same length as THOs and were attached with a phosphate group at the3′ end. The nucleotide sequence for HPV16 PCO is: 5′GGGTTGCTTCTGTGAGTCTTGAA 3′. The nucleotide sequence for HPV18 PCO is: 5′GGTTAACTGGAGTTTTATTATGAGGCT 3′. A THO and PCO were combined at a ratioof 1:2 to form a partially double-stranded nucleic acid probe.

Targets were prepared by PCR amplification of the HPV16 and HPV18sequences and cloned into a pJet vector (Clonejet PCR cloning kit,Fermentas). Plasmids were purified with Genejet plasmid miniprep kit(Fermentas). Forward and reverse primers were designed to be upstreamand downstream of the probe-binding region. The sequences for HPV16forward and reverse primers are 5′ AGACATTTTATGCACCAAAAGAGAACT 3′ and 5′TCTGTGCATAACTGTGGTAACTTTCTG 3′, respectively. The sequences for HPV18forward and reverse primers are 5′ GTATGCATGGACCTAAGGCAACA 3′ and 5′TCGCTTAATTGCTCGTGACATAGA 3′, respectively. Oligonucleotides weresynthesized by Eurogentec.

PCR reactions in a final volume of 25 ul consist of two equal amounts ofmix: 12.5 ul of 2× FastStart Universal Probe master (Rox) (RocheDiagnostics Gmbh, Mannheim Germany) and 12.5 ul primer/probe mix.Primer/probe mix was created as follows: the primers and probes weremixed to a final concentration of 0.4 μM of probes and 0.6 μM ofprimers, and various amounts of target templates were added.Amplification reactions and melting profiles were performed in aStratagene real-time PCR MX3005P system. The thermal profile was: 95° C.for 9 min 30 sec; 40 cycles of 95° C. for 20 sec and 60° C. for 60 sec.Fluorescence measurements were recorded during the read steps at 60° C.Post-amplification melting profile had the following conditions: afterthe last cycle of PCR, heat at 95° C. for 10 sec, cool to 30° C. andhold for 30 sec, then slowly increase the temperature to 80° C. Thefluorescence emission data is continually collected during the risingtemperatures. The first negative derivative of the emission reading,with respect to temperature, is plotted against the temperature to formmelting curves, and the peak of the curve corresponds to the T_(m) ofthe probe.

Sequence Listing Free Text

SEQ ID NOs: 1, 2, 5, 7 and 21 <223> K10 derived PCR primerSEQ ID NOs: 3, 4, 6 and 8-10 <223> SV40 derived PCR primerSEQ ID NOs: 11-16 <223> Jak2 derived PCR primerSEQ ID NOs: 17-20 <223> Kras derived PCR primer

1. A method for assaying a sample for one or more target nucleic acids,said method comprising: (a) contacting a sample comprising one or moretarget nucleic acids with an amplification reaction mixture comprising:(i) one or more pairs of forward/reverse oligonucleotide primers,wherein the primer pairs are capable of amplifying one or more targetnucleic acids, if present in the sample, (ii) a set of two or moreprobes, wherein at least one probe in the set comprises adouble-stranded portion, wherein at least one probe comprises twooligonucleotides: first oligonucleotide, which is also referred to as atarget-hybridising oligonucleotide (THO) and second oligonucleotide,which is also referred to as a partially complementary oligonucleotide(PCO), THO and PCO are capable of hybridising to each other, forming apartially double-stranded probe, wherein each probe in the set comprisesa detectable label or detectable combination of labels which is/arecapable of producing a changeable signal which is characteristic foreach probe, and wherein said two or more probes comprise the samedetectable label or different detectable labels with undistinguishableemission spectra and wherein the melting characteristics of each of suchprobes are different, each probe with double-stranded portion has asignature melting temperature, whereas single-stranded probes having nodouble stranded portion do not have a signature melting temperature; (b)performing an amplification reaction on the sample/reaction mixtureunder amplification conditions, wherein, when a target nucleic acid ispresent, THO which are substantially complementary to part of thattarget nucleic acid are hybridised with the target nucleic acid,therefore being consumed, wherein the consumption of probes causeschanges of detectable signal in the labels, and the consumed probes areno longer able to form a double stranded portion if the original probehas a double-stranded portion; and (c) measuring, at least once, themelting profile of the unconsumed probes in the reaction mixture bydetecting the signal(s) from the labels in those probes as a function oftemperature, wherein the presence or absence of melting characteristicsof any probes in the melting profile analysis is an indication ofunconsumption or consumption of that probe, which further provides anindication of whether or not at least one target nucleic acid is presentin said sample, wherein THO is complementary to a target sequence, andis labeled with a fluorophore and a quencher, wherein PCO, which ispartially complementary to THO, contains modified the 3′ end to preventits extension, e.g. by attaching a label or a phosphate group, wherein,if the 3′ end of PCO is attached with a label which is not quencher, forexample a phosphate group, fluorescence emission is increased byhybridisation of THO and PCO, this type of probe is termed plus probe(+THO:PCO), wherein, if the 3′ end of PCO is attached with a quencher,fluorescence emission is decreased by hybridisation of THO and PCO, thistype of probe is termed minus probe (−THO:PCO).
 2. A method according toclaim 1, wherein said set of two or more probes comprises plus probesonly.
 3. A method according to claim 1, wherein said set of two or moreprobes comprises minus probes only.
 4. A method according to claim 1,wherein said set of two or more probes comprises mixed plus probes andminus primers.
 5. A method according to claim 1, wherein said set of twoor more probes comprises mixed single-stranded probe and plus probes orminus probes.
 6. A method according claim 1, wherein said meltingprofile is measured before reaction/amplification takes place(pre-amplification melting profile), and/or is measured after completionof reaction/amplification (post-amplification melting profile), and/oris measured during reaction/amplification at each cycle or selectedcycles (mid-amplification melting profile), wherein said methodadditionally comprises step (d) (i) comparing at least two meltingprofiles obtained in (c) and/or (ii) comparing a melting profileobtained in step (c) with a previously-obtained melting profile of thesame probes or with a melting profile of the same probes obtained inparallel at the same time in control reactions, or with a theoreticalmelting profile of the same probes wherein a change in the meltingprofile provides an indication of whether or not at least one targetnucleic acid is present in said sample/reaction mixture, wherein saidpre-amplification melting profile is measured in the same reactionvessel before the start of reaction/amplification, or is measured in aseparate reaction vessel where no amplification takes place due to thatreaction mixture lacking one or more ingredients necessary for thereaction/amplification, wherein in step (d) the post-amplification ormid-amplification melting profile is compared with the pre-amplificationmelting profile of the duplex of probes to determine whether aparticular probe is consumed, this being indicative of the presence ofthe corresponding target in the sample.
 7. A method according to claim1, wherein said consumption of probes is achieved through hybridisationof the THO to the target sequence, which is followed by theincorporation of the THO into the amplified product, or wherein when theTHO can be incorporated into the amplified product, the THO is anextendable primer or one of the pair of forward/reverse oligonucleotideprimers.
 8. A method according to claim 1, wherein said consumption ofprobes is achieved through hybridisation of the THO to the targetsequence, which is followed by degradation of the THO, wherein when theTHO is degraded during the reaction, the reaction mixture comprises anenzyme with nuclease activity.
 9. A method according to claim 1, whereinsaid set of two or more probes comprises at least two probes havingdouble-stranded portions, wherein a first probe has a meltingtemperature T_(m)1 in terms of its double-stranded portion, wherein asecond probe has a melting temperature T_(m)2 in terms of itsdouble-stranded portion, wherein T_(m)1>T_(m)2, wherein the same labelsare independently attached to the first and second probes, wherein areduction of any melting peak at T_(m)1 and/or T_(m)2 providesindication of consumption of the first and/or second probe(s).
 10. Amethod according to claim 1, wherein said amplification reaction mixturecomprises two related THO (probes) for assaying single nucleotidepolymorphism (SNP), one THO is for one allele, second THO is for secondallele.
 11. A method according to claim 10, wherein said two relatedTHOs for assaying single nucleotide polymorphism (SNP) are capable ofhybridising the same PCO to form partially double stranded probes.
 12. Amethod according to claim 10, wherein said two related THOs are attachedwith the same label(s).
 13. A method according to claim 1, wherein thesecond oligonucleotides of the probes is physically separated from themain reaction mix containing first oligonucleotides of the probe,primers, reaction buffer, enzyme and other ingredients necessary for areaction during the amplification, but is mixed with the main reactionmix after the completion of the amplification process to measure themelting profile, wherein the second oligonucleotides are added to themain reaction vessel after the reaction completion.
 14. A kit forassaying for one or more nucleic acid targets, which comprises a set oftwo or more probes comprising: at least one probe in the set comprises adouble-stranded portion, wherein said at least one probe comprises twooligonucleotides: a target-hybridising oligonucleotide (THO) and apartially complementary oligonucleotide (PCO), THO and PCO are capableof hybridising to each other, forming a partially double-stranded probe,wherein each probe in the set comprises a detectable label or detectablecombination of labels which is/are capable of producing a changeablesignal which is characteristic for each probe, and wherein said two ormore probes comprise the same detectable label or different detectablelabels with undistinguishable emission spectra and wherein the meltingcharacteristics of each of such probes are different, each probe with adouble-stranded portion has a signature melting temperature, whereassingle-stranded probes having no double stranded portion do not have asignature melting temperature; wherein THO is complementary to a targetsequence, and is labeled with a fluorophore and a quencher, wherein PCO,which is partially complementary to THO, contains modified the 3′end toprevent its extension, e.g. by attaching a label or a phosphate group,wherein, if the 3′ end of PCO is attached with a labels which is not aquencher, for example a phosphate group, fluorescence emission isincreased by hybridisation of THO and PCO, this type of probe is termedplus probe (+THO:PCO), wherein, if the 3′ end of PCO is attached with aquencher, fluorescence emission is decreased by hybridisation of THO andPCO, this type of probe is termed minus probe (−THO:PCO), wherein saidset of two or more probes comprises plus probes only, or said set of twoor more probes comprises minus probes only, or said set of two or moreprobes comprises mixed plus probes and minus primers. or said set of twoor more probes comprises mixed single-stranded probe and plus probes orminus probes.
 15. A kit according to claim 14, wherein said set of twoor more probes comprises two related THOs (probes) for assaying singlenucleotide polymorphism (SNP), one THO is for one allele, second THO isfor second allele., wherein said two related THOs are capable ofhybridising the same PCO to form partially double stranded probes,wherein said two related THOs are attached with the same label(s).